Hemoglobin contrast in magneto-motive optical doppler tomography, optical coherence tomography, and ultrasound imaging methods and apparatus

ABSTRACT

Provided herein are systems, methods, and compositions for the use of optical coherence tomography for detection of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 11/550,771, filed Oct. 18, 2006, is acontinuation-in part of application Ser. No. 11/441,824, filed May 26,2006, which claims the benefit of U.S. Provisional Application No.60/685,559, filed on May 27, 2005, and is a continuation-in-part ofapplication Ser. No. 11/784,477, filed Apr. 6, 2007, which claims thebenefit of U.S. Provisional Application No. 60/790,248, filed Apr. 7,2006. The aforementioned applications are herein incorporated byreference in their entirety.

FUNDING

This invention was supported by funds from the National Institutes ofHealth (AR47551, EB002495 and EB002021) and the Texas AdvancedTechnology Program. The U.S. Government may have certain rights in theinvention.

BACKGROUND

The present invention relates in general to the art of medicaldiagnostic imaging and in particular to imaging blood flow usingmagneto-motive optical Doppler tomography (MM-ODT), Optical CoherenceTomography, or Ultrasound, which combines an externally appliedtemporally oscillating high-strength magnetic field with ODT, OCT, orUltrasound to detect erythrocytes moving according to the fieldgradient.

The accurate determination of location and flow velocity of movingparticles in highly scattering media, such as blood flow, is importantfor medical diagnostics. While the measurements of blood flow in thecoronary arteries is an important aspect in diagnosing coronary arterydiseases. Numerous non-invasive approaches have been developed usingtechniques such as Doppler ultrasound, conventional angiography, laserDoppler flowmetry and magnetic resonance angiography.

One common sensing technique involves the use of ultrasound. Using thistechnique, ultrasound is directed into the body of the patient and tinyparticles such as red blood cells, which are suspended in the bloodplasma, scatter the ultrasonic energy back towards the receiver ortransducer. The transducer then converts the back-scattered ultrasonicenergy into an electrical signal that is processed in some known mannerto determine the presence of a flow and an estimate of the flowvelocity.

Magnetic resonance imaging (MRI) is based on an imaging technique formagnetically exciting nuclear spins in a subject positioned in a staticmagnetic field by applying a radio-frequency (RF) signal of the Larmorfrequency, and reconstructing an image using MR signals induced by theexcitation. MRI is widely applied in clinical medicine because of itscapability of clearly depicting the slightest tissue of human brain invivo.

Magnetic resonance angiography (MRA) provides detailed angiographicimages of the body in a non-invasive manner. In conventional MRA, whichdoes not use contrast agents, magnetic resonance signal from flowingblood is optimized, while signal from stationary blood or tissuestructures is suppressed. In contrast-enhanced MRA, a contrast agent isinjected into the blood stream to achieve contrast between flowing bloodand stationary tissue.

The commonly known echo planar imaging (EPI) is a rapid MRI technique,which is used to produce tomographic images at high acquisition rates,typically several images per second. Functional magnetic resonanceimaging (fMRI) has been found useful in perfusion and/or diffusionstudies and in dynamic-contrast studies, etc. However, images obtainedin EPI experiments tend to be vulnerable to an artifact known as“ghosting” or “ghost images.”

Optical coherence tomography (OCT) is a technology that allows fornon-invasive, cross-sectional optical imaging of biological media withhigh spatial resolution and high sensitivity. OCT is an extension oflow-coherence or white-light interferometry, in which a low temporalcoherence light source is utilized to obtain precise localization ofreflections internal to a probed structure along an optic axis. Thistechnique is extended to enable scanning of the probe beam in thedirection perpendicular to the optic axis, building up a two-dimensionalreflectivity data set, used to create a cross-sectional gray-scale orfalse-color image of internal tissue backscatter.

OCT uses the short temporal coherence properties of broadband light toextract structural information from heterogeneous samples such asbiologic tissue. OCT has been applied to imaging of biological tissue invitro and in vivo. Systems and methods for substantially increasing theresolution of OCT and for increasing the information content of OCTimages through coherent signal processing of the OCT interferogram datahave been developed to provide cellular resolution (i.e., in the orderof 5 micrometers). During the past decade, numerous advancements in OCThave been reported including real-time imaging speeds.

In diagnostic procedures utilizing OCT, it would also be desirable tomonitor the flow of blood and/or other fluids, for example, to detectperipheral blood perfusion, to measure patency in small vessels, and toevaluate tissue necrosis. Another significant application would be inretinal perfusion analysis. Accordingly, it would be advantageous tocombine Doppler flow monitoring with the above micron-scale resolutionOCT imaging in tissue.

Conventional OCT imaging primarily utilizes a single backscatteringfeature to display intensity images. Functional OCT techniques processthe backscattered light to provide additional information onbirefringence, and flow properties. (See for example, Kemp N J, Park J,Zaatar H N, Rylander H G, Milner T F, High-sensitivity determination ofbirefringence in turbid media with enhanced polarization-sensitiveoptical coherence tomography, Journal of the Optical Society of AmericaA: Optics Image Science and Vision 2005, 22(3):552-560; Dave D P, AkkinT, Milner T E, Polarization-maintaining fiber-based opticallow-coherence reflectometer for characterization and ranging ofbirefringence, Optics Letters 2003, 28(19): 1775-1777; Rylander C G,Dave D P, Akkin T, Milner T E, Diller K R, Welch M, Quantitativephase-contrast imaging of cells with phase-sensitive optical coherencemicroscopy, Optics Letters 2004, 29(13):1509-1511; de Boer J F, Milner TE, Ducros M G, Srinivas S M, Nelson J S, Polarization-sensitive opticalcoherence tomography, Handbook of Optical Coherence Tomography, NewYork: Marcel Dekker, Inc., 2002, pp 237-274.)

Since the ability to characterize fluid flow velocity using OCT wasdemonstrated by Wang et al., several phase resolved, real-time opticalDoppler tomography (ODT) approaches have been reported. (See forexample, Chen Z P, Milner T E, Dave D, Nelson J S, Optical Dopplertomographic imaging of fluid flow velocity in highly scattering media,Optics Letters 1997, 22(1):64-66; Wang X J, Milner T E, Nelson J S.

Optical Doppler tomography (ODT) combines Doppler velocimetry withoptical coherence tomography (OCT) for noninvasive location andmeasurement of particle flow velocity in highly scattering media withmicrometer-scale spatial resolution. The principle employed in ODT isvery similar to that used in radar, sonar and medical ultrasound. ODTuses a low coherence or broadband light source and opticalinterferometer to obtain high spatial resolution gating with a highspeed scanning device such as a conventional rapid scanning opticaldelay line (RSOD) to perform fast ranging of microstructure and particlemotion detection in biological tissues or other turbid media.

To detect the Doppler frequency shift signal induced by the movingparticles, several algorithms and hardware schemes have been developedfor ODT. The most straightforward method to determine the frequencyshift involves the use of a short time fast Fourier transform (STFFT).However, the sensitivity of this method is mainly dependent on the FFTtime window, which limits axial scanning speed and spatial resolutionwhen measuring slowly moving blood flow in small vessels that requireshigh velocity sensitivity. However, a phase-resolved technique candecouple the Doppler sensitivity and spatial resolution whilemaintaining high axial scanning speed.

In ODT, the Doppler frequency shift is proportional to the cosine of theangle between output and input scattering directions of the probe beamand the scatterer's flow direction. When the two directions areperpendicular, the Doppler shift is zero. Because a priori knowledge ofthe Doppler angle is not available, and conventional intensity OCTimaging provides a low contrast image of microvasculature structure,detecting small vessels with slow flow rates is difficult. However, theDoppler angle can be estimated by combining Doppler shift and Dopplerbandwidth measurements. (See for example, Piao D Q, Zhu Q, QuantifyingDoppler Angle and Mapping Flow Velocity by a Combination ofDoppler-shift and Doppler-bandwidth Measurements in Optical DopplerTomography, Applied Optics, 2003, 42(25): 5158-5166, and U.S. Pat. No.5,991,697 describe a method and apparatus for Optical DopplerTomographic imaging of a fluid flow in a highly scattering mediumcomprising the steps of scanning a fluid flow sample with an opticalsource of at least partially coherent radiation through aninterferometer, which is incorporated herein by reference).

The ability to locate precisely the microvasculature is important fordiagnostics and treatments requiring characterization of blood flow.Recently, several efforts to increase blood flow contrast mechanismshave been reported including protein microspheres incorporatingnanoparticles into their shells, plasmon-resonant gold nanoshells, anduse of magnetically susceptible micrometer sized particles with anexternally applied magnetic field. (See for example, Lee T M, OldenburgA L, Sitafalwalla S, Marks D L, Luo W, Toublan F J J, Suslick K S,Boppart S A, Engineered microsphere contrast agents for opticalcoherence tomography, Optics Letters, 2003, 28(17): 1546-1548; Loo C,Lin A, Hirsch L, Lee M H, Barton J, Halas N, West J, Drezek R.Nanoshell-enabled photonics-based imaging and therapy of cancer.Technology in Cancer Research & Treatment, 2004; 3(1): 33-40; andOldenburg A L, Gunther J R, Boppart S A, Imaging magnetically labeledcells with magnetomotive optical coherence tomography, Optics Letters,2005, 30(7): 747-749.)

Wang, et al., “Characterization of Fluid Flow Velocity by OpticalDoppler Tomography,” Optics Letters, Vol. 20, No. 11, Jun. 1, 1995,describes an Optical Doppler Tomography system and method which usesoptical low coherence reflectometry in combination with the Dopplereffect to measure axial profiles of fluid flow velocity in a sample. Adisadvantage of the Wang system is that it does not provide a method todetermine direction of flow within the sample and also does not providea method for generating a two-dimensional color image of the sampleindicating the flow velocity and directions within the image.

The use of an externally applied field to move magnetically susceptibleparticles in tissue has been termed magneto-motive OCT (MM-OCT).Functional magnetic resonance imaging (fMRI) detects deoxyhemoglobinwhich is a paramagnetic molecule. However, the paramagneticsusceptibility of human tissue is very low compared to otherbiocompatible agents such as ferumoxides (nanometer sized iron oxideparticles). Therefore, it was believed that, other than differentiatingrelaxation times (T2) between oxygenated and deoxygenated blood, themagnetic field strength required to produce a retarding force on bloodflow was well above that of current imaging fields. (See also, forexample, Schenck J F., Physical interactions of static magnetic fieldswith living tissues, Progress in Biophysics and Molecular Biology 2005,87 (2-3): 185-204; and Taylor D S, Coryell, C. D., Magneticsusceptibility of iron in hemoglobin. J. Am. Chem. Soc. 1938,60:1177-1181.)

SUMMARY OF THE INVENTION

The invention is a method and apparatus of imaging a blood flow,hemoglobin, and/or nanoparticles using optical coherence tomography,which comprises an externally applied temporally oscillatinghigh-strength magnetic field with an optical tomography system to detecthemoglobin and/or nanoparticles.

Another embodiment is an apparatus for imaging blood flow and/ornanoparticles, comprising a magnetomotive optical Doppler tomography(MM-ODT) imaging system

Another aspect of the invention is a method for imaging a blood flow,comprising applying a magnetic field to the blood flow, wherein theblood flow comprising a plurality of hemoglobin molecules and/ornanoparticles and wherein the magnetic field interacts with thehemoglobin molecules to cause a change in the blood flow; and detectingthe blood flow by detecting the change in the blood flow caused by theinteraction with the hemoglobin molecules, wherein the change isdetected using a magnetomotive optical Doppler tomography imagingsystem.

In another embodiment, the invention comprises a solenoid cone-shapedferrite core with an extensively increased magnetic field strength atthe tip of the core. In a further embodiment, the invention comprisesfocusing the magnetic force on targeted samples.

Another embodiment of the method for imaging a blood flow comprisestemporally oscillating the magnetic field.

Another embodiment of the invention is an apparatus for imaging a bloodflow comprising a magnetic field generator for applying a magnetic fieldto the blood flow, wherein the blood flow comprises hemoglobin moleculesand/or nanoparticles; and an ultrasound detection system for detectingthe blood flow while it is in the presence of the magnetic field.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate aspects of the methods,apparatuses, and systems and together with the description, serve toexplain the principles of the methods, apparatuses, and systems.

FIG. 1 is a schematic diagram of the MM-ODT system.

FIG. 2 is a schematic diagram of the probe beam, flow sample andsolenoid coil.

FIGS. 3A and 3B are OCT and ODT images of a stationary turbid solutionwithout an external magnetic field, respectively. FIG. 3C and FIG. 3Dare OCT and ODT images with a 50 Hz magnetic field, respectively. Thewhite bar represents 200 μm, accordingly.

FIGS. 4A-4D are M-mode ODT images of the diluted deoxygenated blood flow(18% hematocrit) without and with an external magnetic field. FIG. 4Aand FIG. 4B are ODT images of 5 mm/s blood flow without and with a 5 Hzmagnetic field, respectively. FIG. 4C and FIG. 4D are ODT images of 30mm/s blood flow without and with a 50 Hz magnetic field, respectively.The Black bar indicates 200 μm, accordingly.

FIG. 5A and FIG. 5B are the Doppler frequency shift profiles without anexternal magnetic field, and with a 50 Hz magnetic field, respectively.

FIG. 6 is a block diagram illustrating an exemplary phase sensitive OCTsystem.

FIG. 7 is a graph of the optical path length change in the DP-OCTsystem.

FIG. 8 is a schematic diagram of Magneto Motive Ultrasound for Blood.

FIG. 9 is an ultrasound image of blood exposed to an oscillatingmagnetic field.

FIG. 10 is a schematic diagram of Ultrasonography and OCT coupled withmagnetic field generator.

FIGS. 11A and 11B show solenoid drive signal and optical pathlengthchange observed in a mouse imaged with metallic nanoparticles (11A) anda mouse imaged without metallic nanoparticles (11B).

FIGS. 12A-B shows a schematic diagram of a differential phase opticalcoherence tomography (DP-OCT) system combined with a magnetic fieldgenerator: (12A) DP-OCT system, (12B) collinear configurations of theDP-OCT sample path and design of the magnetic field generator containinga conical iron core.

FIG. 13A-B shows the optical path length change (Δp) in livers withdifferent SPIO doses (1.0, 0.1 mmol Fe/kg and saline control) usingfocused magnetic field excitation (2 Hz, 4 V_(pp)) (13A). Optical pathlength change (Δp) in specimens with doses 1.0 mmol Fe/kg SPIO (13B),0.1 mmol Fe/kg SPIO, and a saline control liver. The applied magneticflux density strength is B_(z)=0.47 Tesla at the liver specimen.

FIG. 14A-B shows the maximum optical path length change (Δp) iniron-laden liver specimens due to nanoparticle movement in response to afocused magnetic field for mice injected with various SPIO doses (1.0and 0.1 mmol Fe/kg). The input frequency is 2 Hz with applied voltageranging from 2 to 8 V_(pp) (14A) and magnetic field strength at eachinput voltage (14B).

FIG. 15A-D shows the Optical path length change (Δp) in iron-laden liverspecimens due to nanoparticle movement in response to a focused magneticfield with a swept frequency (1˜10 Hz) input for mice injected withvarious SPIO doses (1.0 and 0.1 mmol Fe/kg). (15A). Optical path lengthchange (Δp) at 1.0 mmol Fe/kg SPIOdose (15B), 0.1 mmol Fe/kg SPIO dose(15C), and a saline control liver (15D). The applied focused magneticflux density is 1.3 Tesla at the specimen.

FIGS. 16A-B shows the maximum optical path length change (Δp) iniron-laden liver specimens due to nanoparticle movement in response to afocused magnetic field with a swept frequency (1˜10 Hz) input for miceinjected with various SPIO doses (1.0 and 0.1 mmol Fe/kg). Input sweptfrequency ranged from 1˜10 Hz over 2 seconds with input voltagesincreasing from 2 to 10 V_(pp) (16A) and magnetic field strength at eachinput voltage (16B).

FIGS. 17A-D shows optical path length change (Δp) in iron-laden rabbitarteries (0.1 Fe/kg) measured in response to 2 Hz frequency sinusoidalinputs.

DETAILED DESCRIPTION OF THE INVENTION

The methods, apparatuses, and systems can be understood more readily byreference to the following detailed description of the methods,apparatuses, and systems and the Examples included therein and to theFigures and their previous and following description.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theseare not limited to specific synthetic methods, specific components, orto particular compositions, as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a nanoparticle”includes mixtures of nanoparticles, reference to “a nanoparticle”includes mixtures of two or more such nanoparticles, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally substituted shelledmetals” means that shelled metals may or may not be substituted and thatthe description includes both unsubstituted shelled metals and shelledmetals where there is substitution.

As used throughout, by a “subject” is meant an individual. Thus, the“subject” can include domesticated animals, such as cats, dogs, etc.,livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds. In oneaspect, the subject is a mammal such as a primate or a human,alternatively, cats, dogs, and other livestock may be used for testingpurposes. The term does not denote a particular age or sex. Thus, adultand newborn subjects, as well as fetuses, whether male or female, areintended to be covered.

It is to be understood that nanoparticle and/or hemoglobin may beaffected by an external oscillating magnetic field for magneto-motiveOptical Coherence Tomographic imaging.

Reference will now be made in detail to exemplary aspects of thesystems, methods, apparatuses, and/or compositions, examples of whichare illustrated in the accompanying drawings.

Provided herein are methods, compositions and apparatuses forMagneto-Motive Optical Doppler Tomography (“MM-ODT”) for improvedDoppler imaging of blood flow and/or nanoparticles using an externaloscillating magnetic field is described below. By introducing mechanicalmovement of red blood cells (RBC's) or nanoparticles by a temporallyoscillating high-strength magnetic field, MM-ODT allows imaging of bloodflow and velocity. The controlled and increased Doppler frequency inMM-ODT provides an investigational tool to study in vivo bloodtransport, as shown in the article Hemoglobin Contrast in MagnetomotiveOptical Doppler Tomography, Opt. Lett. 31, 778-780 (2006), hereinincorporated by reference.

The microstructure of the blood flow and flow velocity information areall encoded in the interferogram of a Doppler OCT system. It should bereadily apparent to those skilled in the optical arts, that differentOCT systems and different OCT information can be used to determine theDoppler frequency shift. It is not intended to suggest any limitation asto the scope or functionality with different OCT architectures oroptical information used with an oscillating magnetic field, such astime domain Doppler OCT and spectral domain Doppler OCT. Time domain OCTrequires a mechanism that varies the pathlength of light propagating inthe reference path of the interferometer, while spectral domain OCTincludes swept source OCT and Fourier domain OCT. An example oftime-domain Doppler OCT is provided below.

A schematic of the MM-ODT apparatus 10 is shown in FIG. 1. The OCT lightsource 12 comprises a super luminescent diode, which is used as the lowcoherence light source, in one embodiment of the invention, where thelight source 12 is centered at 1.3 μm with a bandwidth of 90 nm.Alternatively, a tunable laser source in the spectral domain may be usedas the light source 12. Light from source 12 is coupled into asingle-mode optical fiber based interferometer 20 by circulator 14,where the interferometer 20 can provide 1 mW of optical power at thesample 60. Light is split into a reference arm 22 and sample arm 24 by a2×2 splitter 16. A rapid-scanning optical delay (RSOD) line 26 iscoupled to the reference arm 22. In one embodiment, the rapid-scanningoptical delay line 26 is aligned such that no phase modulation isgenerated when the group phase delay is scanned at ˜4 kHz. In the samplearm 24, a collimated beam 36 is redirected to sample 60 by twogalvanometers 30 that permit three-dimensional scanning. In oneembodiment, the galvanometers can be an X-scanner and a Y-scanner. Thesample 60 can be internal or external to the body, where the probe beamis focused by an objective lens 32. In one embodiment, the objectivelens 32 yields a 10-μm spot at the focal point. Phase modulation can begenerated using an electro-optical waveguide phase modulator, which canproduce a carrier frequency (˜1 MHz). And the magnetic field generator62 is in proximity to the sample 60.

A dual-balanced photodetector 34 is coupled to the 2×2 splitter 16 andthe circulator 14. The photodetector 34 of an 80 MHz bandwidth reducesthe light source noise from the OCT interference signal. A hardwarein-phase and quadrature demodulator 40 with high/bandpass filters 42 andlow/bandpass filters 44 improves imaging speed. Doppler information wascalculated with the Kasai autocorrelation velocity estimator. Labviewsoftware 50 (National Instruments, Austin, Tex.) is coupled to theMM-ODT system with a dual processor based multitasking scheme. Themaximum frame rate of the MM-ODT system 10 was 16 frames per second fora 400×512 pixel sized image. The Doppler frequency shift can bedetermined with the use of a short time fast Fourier transform (STFFT).Alternatively, a phase-resolved technique can determine the Dopplerfrequency shift to decouple the Doppler sensitivity and spatialresolution while maintaining high axial scanning speed. Alternatively,differential phase optical coherence tomography (OCT) or spectral domainphase-sensitive OCT can be used to determine the Doppler frequencyshift, as readily determined by one skilled in the optical arts.

FIG. 2 shows an example of the magnetic field generator 80 with acapillary tube 90. The magnetic field generator 80 includes a solenoidcoil 82 (Ledex 4EF) with a cone-shaped ferrite core 84 at the center anddriven by a current amplifier 86 supplying up to 960 W of power. Themagnetic field generator 80 can be placed underneath the sample 60during MM-ODT imaging. The combination of the ferrite core 84 andsolenoid coil 82 using a high power operation dramatically increases themagnetic field strength (B_(max)=0.14 Tesla) at the tip of the core 82and also focuses the magnetic force on the targeted samples 60. Thesinusoidal current can vary the magnetic force applied to the capillarytube 90 in order to introduce movement of magnetic fluids, which includered blood cells that contain hemoglobin. In one embodiment, the probebeam is oriented parallel to the gradient of the magnetic field'sstrength.

The material parameter characterizing magnetic materials, includingbiological tissue, is the magnetic volume susceptibility, χ. Magneticvolume susceptibility is dimensionless in SI units and is defined by theequation M=χH, where M is the magnetization at the point in question andH is the local density of the magnetic field strength. Hemoglobin's highiron content, due to four Fe atoms in each hemoglobin molecule, and thelarge concentration of hemoglobin in human red blood cells allowHemoglobin magneto-motive effects in biological tissue. The magneticvolume susceptibility of the hemoglobin molecule consists of aparamagnetic component due to the electron spins of the four iron atoms.The paramagnetic susceptibility is given by the Curie Law,

$\begin{matrix}{\chi = \frac{\mu_{o}{N_{p}\left( {\mu_{eff}^{2}\mu_{B}^{2}} \right)}}{3{kT}}} & (1)\end{matrix}$

where μ_(o) is permeability of free space and has the value 4π×10 ⁷ H/m,N_(p) is the volume density of paramagnetic iron atoms in hemoglobin,N_(p)=4.97×10²⁵ iron atoms/m³, μ_(eff) is the effective number of Bohrmagnetons per atom reported as 5.35, and the Bohr magneton,μ_(B)=9.274×10⁻²⁴ J/T, and Boltzmann's constant, k=1.38×10⁻²³ J/K, and Tis the absolute temperature (K). The calculated susceptibility of a RBCis about 11×10⁻⁶ assuming a 90% concentration of hemoglobin per RBC. Thecalculated susceptibility of a RBC is dependent on the oxygenation ofthe hemoglobin. The calculations can be adjusted accordingly, dependingon the oxygenation of the RBC, which can be measured by knowntechniques.

A RBC placed in a magnetic field gradient experiences forces and torquesthat tend to position and align it with respect to the field'sdirection. The magnetic force, in the direction of the probing light z,is given by

$\begin{matrix}{{F_{z} = {{m_{RBC}\frac{\delta^{2}{z(t)}}{d\; t^{2}}} = {\frac{\delta \; U}{\delta \; z} = {\frac{\Delta \; \chi \; V}{\mu_{o}}B\frac{\delta \; B}{\delta \; z}}}}},} & (2)\end{matrix}$

where V is the particle volume, B is the magnitude of the magnetic fluxdensity, and Δχ is the difference between the susceptibility of theparticle and the surrounding medium. The displacement [z(t)] of an RBCdriven by a time varying magnetic flux density can be included in theanalytic OCT fringe expression, I_(f),

$\begin{matrix}{{I_{f} = {2\sqrt{I_{r}I_{s}}{\exp \left\lbrack {i\left( {{2\pi \; f_{o}t} + {\frac{4\pi \; {z(t)}}{\lambda_{o}}{z(t)}}} \right)} \right\rbrack}}},} & (3)\end{matrix}$

where I_(R) and I_(S) are the back scattered intensities from thereference and sample arms, respectively, f_(o) is the fringe carrierfrequency, λ_(o), is the center wavelength of the light source, and z(t)is the RBC displacement. Integration of all forces (magnetic, elastic,and viscous) on the RBC gives a steady state displacement, z(t)=Acos(4πf_(m)t) where A is a constant in units of length and f_(m) is themodulation frequency of the magnetic flux density. In free space, thedisplacement, z(t), is dominated a constant acceleration which can be,however, ignored in confined models (i.e. blood vessel or capillarytube) with assumptions that, first, the probing area is much smallerthan magnetic field area, and that secondly probing time starts aftersteady states of inner pressures. Expansion of the right-hand side ofEq. (3) using Bessel functions gives

$\begin{matrix}{I_{f}\alpha \; 2\sqrt{I_{R}I_{S}}\left( {{\sum\limits_{k = 0}^{\infty}\left( {{J_{k}(m)}{\exp \left( {\; {k4}\; \pi \; f_{m}t} \right)}} \right)} + {\sum\limits_{k = 0}^{\infty}\left( {\left( {- 1} \right)^{k}{J_{k}(m)}{\exp \left( {{- }\; k\; 4p\; f_{m}t} \right)}} \right)}} \right){\exp\left( {\; 2\pi \; f_{o}t} \right.}} & (4)\end{matrix}$

where J_(k)(m) is the Bessel function of the first kind of order k forargument m which is 4πA/λ_(o). The amplitude of the k^(th) sideband isproportional to J_(k)(m) In coherent detection, the fraction of opticalpower transferred into each of the first order sidebands is (J_(l)(m))²,and the fraction of optical power that remains in the carrier is(J_(o)(m))².

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompositions, compositions, articles, devices, systems, and/or methodsclaimed herein are made and evaluated, and are intended to be purelyexemplary and are not intended to limit the scope of compositions,compositions, articles, devices, systems, and/or methods. Efforts havebeen made to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.), but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in degrees Celsius or is at ambient temperature, and pressure is ator near atmospheric.

Example 1 MM-ODT Imaging of the Doppler Shift of Hemoglobin by Applyingan Oscillating Magnetic Field to a Moving Blood Sample

M-mode OCT/ODT images of a capillary glass tube filled with a stationaryturbid solution with and without an external magnetic field as a controlsample were recorded, as shown in FIGS. 3-4. A 750 μm-inner diameterglass capillary tube 200 was placed perpendicularly to the probing beam70, as shown in FIG. 2. Fluids used for flow studies were injectedthrough the tube at a constant flow rate controlled by a dual-syringepump (Harvard Apparatus 11 Plus, Holliston, Mass.) with ±0.5% flow rateaccuracy. The turbid solution was a mixture of deionized water and0.5-gm latex microspheres (μ_(s)=5 mm⁻¹). The magnetic flux density andits frequency were approximately 0.14 T and 50 Hz, respectively. M-modeOCT/ODT images were acquired for 100 ms per frame. FIGS. 3 a and 3 bshow M-mode OCT and ODT images without any external magnetic field,respectively. The ODT image in FIG. 3 b contains small random phasefluctuations due to ambient vibration through the optical path. FIGS. 3c and 3 d show M-mode OCT and ODT images with a 50 Hz external magneticfield, respectively. No distinguishable Doppler shift could be observedin the ODT image FIG. 3 d indicating no interaction between the externalmagnetic field and the moving microspheres.

Deoxygenated blood was extracted from the vein of a human male's leftarm, and diluted with saline. During preparation, blood was not exposeddirectly to air so as to remain deoxygenated. To simulate flow, bloodwas injected through the capillary tube 200 by a syringe pump at arelatively constant flow rate. As FIG. 4 shows, the oscillating Dopplerfrequency shift, resulting from RBC movement, could be observed at twodifferent flow rates (5 and 30 mm/s). Because the flow direction wasnearly perpendicular to the probing beam no significant Dopplerfrequency shift was distinguishable at the 5 mm/s flow rate FIG. 4 awithout any external magnetic field. In the case of the high blood flowrate of 30 ml/s, as shown at FIG. 4 c, the Doppler frequency shiftcaused by the blood flow could be observed. And for maximum contrastenhancement, the probe beam can be directed parallel to the gradient ofthe magnetic field's strength. However, application of a 50 Hz magneticfield increased the Doppler contrast of blood at both the slow and fastflow rates as shown at FIGS. 4 b and 4 d. The high flow rate of 30 mm/sgives a higher contrast image than the low flow rate image, but theDoppler frequency shift of the former as a function of depth is lesshomogeneous than the latter, which is indicative of perturbation byblood flow. The same blood was diluted to 5% hematocrit (HCT), but noRBC movement could be observed below 8% HCT.

Doppler frequency shift profiles were calculated from the ODT images byaveraging 20 lines at a selected depth indicated by horizontal arrows,as shown in FIGS. 4 a and 4 b. FIG. 5 a indicates no significant Dopplerfrequency shift over a 100 ms time period, whereas FIG. 5 b displays±200 to 300 Hz Doppler frequency shifts oscillating 20 times over 100 ms(200 Hz).

The Doppler frequency shift indicates that RBC's physically move intoand away from the incident light while passing through the externalmagnetic field depending on whether their magnetic properties areparamagnetic or diamagnetic, as shown in FIG. 4, and that the 200 Hzoscillation of the Doppler frequency shift correlates with the 50 Hzmagnetic field, as shown in FIG. 5. Frequencies 4 times higher than thatof the external magnetic field (f_(m)) can be observed. According to Eq.2, the frequency of the force on paramagnetic targets was twice that ofthe magnetic flux density; therefore, a 50 Hz B field displaces thetargets at 100 Hz. Although the fringe signal (Eq. 4) contains harmonicsat frequency (2f_(m)), the modulation frequency, f_(m), was set so thatthe second-order sideband (4f_(m)) was dominant as shown in FIG. 5 b.The particle motion can not be described as a pure sinusoidal functioneven if the modulated magnetic field is sinusoidal, due to the numerousforces that contribute to the motion within the field such as gravity,concentration gradient, and colloidal dispersion.

The invention is a new investigational tool to study in vivo bloodtransport and the first implementation of MM-ODT for improved Dopplerimaging of blood flow using an external oscillating magnetic fieldintroducing a mechanical movement of RBC's during blood flow by atemporally oscillating high-strength magnetic field. MM-ODT to allowimaging of tissue function in a manner similar to functional magneticresonance images (f-MRI) of deoxygenated blood in organs, when thesample arm of the MM-ODT system is coupled to a probe (not shown). Suchprobes are generally known in the arts, such as endoscopic probes,catheter probes, and the like.

Alternatively, the MM-ODT can be used for Port-Wine vessel mapping andSkin Cancer vessel mapping. The MM-ODT can be used to detect bloodvessel location and size for cancer and port-wine stains, since theseconditions are characterized by blood vessel growth and increases inhemoglobin content. Accordingly, other blood vessel detection for tissueabnormality identification can be envisioned with this invention.Generally, the MM-ODT can be used wherever blood flow detection isnecessary in operations, chemotherapy, hemodialysis, and the like.

A spectral domain phase sensitive OCT system 100 can be used to imagethe blood flow and determine velocity information, when an oscillatingmagnetic field is applied to the blood flow, as shown in FIG. 6.Spectral domain phase sensitive OCT generally uses a broadband lightsource (in a general interferometric setup, where the mirror in thereference typically does not move or does not require a rapid scanningdelay line. A spectral interferogram is detected, in which each A-scanis entirely encoded. A Fourier transformation of the A-scan and velocitydistribution of the blood flow can be extracted by known methods.Spectral domain OCT can be generally understood under U.S. Pat. No.5,991,697, which describes how to measure the Doppler shift. In spectraldomain Doppler OCT, the blood flow profile is the envelope of acalculated A-scan through Fourier transformation on the spectralinterferogram. Velocity information is encoded in the rate of phasechange of the interferogram. The Doppler velocity can be extracted bymeasuring the phase shift between two successive calculated A-scans inspectral domain Doppler OCT and the time between recording successiveA-scans

This OCT system 100 is only an example of one OCT imaging modality whichcan be used to image blood flow with a temporally oscillating magneticfield, and is not intended to suggest any limitation on the scope of OCTarchitectures applicable to the invention. Generally, the OCT system 100includes a general-purpose computing device in the form of a computer101 and includes a magnet control 114 and a magnet 116.

Light energy is generated by a light source 117. The light source 117can be a broadband laser light source coupled into optical fiberemitting light energy over a broad range of optical frequencies. Thewavelength range can be from about 400 nanometers to about 1600nanometers. Longer wavelengths (>800 nm) can be used for deeperscanning. Preferably, the light source emits light having a wavelengthnear the infrared spectrum to identify hemoglobin for OCT imaging, andthe magnet 116 places hemoglobin in motion and increases opticalscattering of the hemoglobin. The light energy can be emitted over amultiplicity of optical wavelengths, frequencies, and pulse durations toachieve OCT imaging. As used herein, optical fiber can refer to glass orplastic wire or fiber. Optical fiber is indicated on FIG. 6 as linesconnecting the various blocks of the figures. Where light energy isdescribed as “passing,” “traveling,” “returning,” “directed,” or similarmovement, such movement can be via optical fiber.

A fraction of the generated light energy passes from the light source117 into an optical spectrum analyzer 118. The optical spectrum analyzer118 measures optical frequency as the light energy is emitted from thelight source 117 as a function of time. The optical spectrum analyzer118 samples a portion of the light emitted by the light source 117. Theoptical spectrum analyzer 118 monitors the power spectral density oflight entering the splitter 119. The remaining fraction of light energyfrom the light source 117 passes into a splitter 119. The splitter 119can be a device with four ports, with Port 1 allowing light energy toenter the splitter 119. Ports 2 and 3 allow light energy to leave andre-enter the splitter 119 to the reference reflector 120 and OCT probe122, respectively. Port 4 allows light energy to leave the splitter 119to coupling lens 124. The splitter 119 couples the light into Port 1.The splitter 119 divides the light according to a pre-determined splitratio selected by a user. For example, the split ratio can be 50/50wherein half of the light energy entering the splitter 119 at Port 1exits the splitter 119 through Port 2 and half exits the splitter 119through Port 3. In another example, the split ratio can be 60/40 wherein60% of the light energy passes through Port 2 and 40% of the lightenergy passes through Port 3.

A fraction of the light energy (determined by the split ratio) thatexits the splitter 119 through Port 2 travels to a reference reflectorsurface 120. The light energy is reflected from the reference reflectorsurface 120 back to the splitter 119 into Port 2. The referencereflector 120 can be a planar metallic mirror or a multilayer dielectricreflector with a specified spectral amplitude/phase reflectivity. Theremaining fraction of light that entered splitter 119 through Port 1exits splitter 119 through Port 3 and enters an OCT probe 122. The OCTprobe 122 can be a turbine-type catheter as described in PatentCooperation Treaty application PCT/US04/12773 filed Apr. 23, 2004 whichclaims priority to U.S. provisional application 60/466,215 filed Apr.28, 2003, each herein incorporated by reference for the methods,apparatuses and systems taught therein. The OCT probe 122 can be locatedwithin a subject 121 to allow light reflection off of subject's 121blood flow.

The light energy that entered OCT probe 122 is reflected off of theblood flow of subject 121 once an oscillating magnetic field has beentemporally applied by magnet 116. The reflected light energy passes backthrough the OCT probe 122 into the splitter 119 via Port 3. Thereflected light energy that is returned into Port 2 and Port 3 of thesplitter 119 recombines and interferes according to the split ratio. Thelight recombines either constructively or destructively, depending onthe difference of pathlengths. A series of constructive and destructivecombinations of reflected light create an interferogram (a plot ofdetector response as a function of optical path length difference). Eachreflecting layer from the subject 121 and the blood flow will generatean interferogram. The splitter 119 can recombine light energy that isreturned through Port 2 and Port 3 so that the light energies interfere.The light energy is recombined in the reverse of the split ratio. Forexample, if a 60/40 split ratio, only 40% of the light energy returnedthrough Port 2 and 60% of the light energy returned through Port 3 wouldbe recombined. The recombined reflected light energy is directed outPort 4 of the splitter 119 into a coupling lens 124. The coupling lens124 receives light from the output of the splitter 119 and sets the beametendue (beam diameter and divergence) to match that of the opticalspectrometer 125. The coupling lens 124 couples the light into anoptical spectrometer 125. The optical spectrometer 125 can divide therecombined reflected light energy light into different opticalfrequencies and direct them to different points in space which aredetected by a line scan camera 126. The line scan camera 126 performslight to electrical transduction resulting in digital light signal data108. The digital light signal data 108 is transferred into the computer101 via the OCT input interface 111. Interface between the line scancamera 126 and computer 101 can be a Universal Serial Bus (USB), or thelike. The digital light signal data 108 can be stored in the massstorage device 104 or system memory 112 and utilized by the imageconstruction software 106 and the Labview image construction software107.

The image construction software 106 can generate an image of the bloodflow of subject 121 from the light signal data 108, by receiving lightsignal data 108 generating amplitude and phase data. The amplitude andphase data (optical path length difference (cτ) or optical time-delay(τ)) can be separated into discrete channels and a plot of intensity vs.depth (or amplitude vs. depth) can be generated for each channel. Suchplot is known as an A-scan, where the composition of all the A-scans cancomprise one image. And movement image construction software 107generates an image of the movement of the hemoglobin from the lightsignal data 108. The movement image construction software 107 receiveslight signal data 108 for at least two successive sweeps of the lightsource 117 and the light source performs a Fourier transform on thelight signal data 108 generating amplitude and phase data.

Optionally, additional information can be extracted from the lightsignal data to generate additional images. The light signal data can befurther processed to generate a Stokes parameter polarimetric image whenused in conjunction with polarization detectors and polarizing splittersto extract polarization data from the light signal 108, as readily knownto one skilled in the art of optical coherence tomography. Thedifferential phase OCT image data is shown in FIG. 7, indicating theoptical path difference with time variation of the magnetic fieldmagnitude. Signal processing filters can be utilized to reduce the noiseof the optical pathlength signal.

Alternatively, the phase sensitive OCT system 100 can be configured forswept source OCT, which is a different type of spectral domain OCT. Inswept source OCT, a tunable laser source replaces the broadband laserlight source 117. The scanning rate can be at wavelengths of 800 nm-1500nm. Also, the reference reflector surface 120 is in-line with samplepath 120. The optical spectrometer 125 and line scan camera 126 arereplaced with a general photodetector. In this configuration, an opticalclock is used to trigger acquisition of the signal produced by thephotodetector. The optical clock provides a set of uniformly spacedclock pulses with fixed intervals of optical frequency and at least onereference pulse. The fixed intervals of optical frequency are configuredand specified in the optical clock to give a uniform train of pulses.The at least one reference pulse generated by the optical clock isutilized to provide a reference optical frequency or a trigger pulse.For example, the first reference pulse generated by the optical clockcan correspond to an absorption line in a gas cell (e.g., HydrogenFluoride or Hydrogen Bromide). In this case the gas absorption line hasa known optical frequency. The well-known absorption fingerprint bandsin the HF gas cell result in a reduced detected intensity in the lighttransmitted through the gas cell, and as such provide a metric on theabsolute lasing wavelength at the digitized samples of the photodetectorsignal. The digitized sample number or sampling time scale can thus beconverted to absolute wavelength at one or more samples, depending onthe number of absorption lines. The detected wavemeter photocurrentsignal and the detected gas cell photocurrent signal are combined in thedigitizer to provide the relationship between the sample number orsampling time and lasing wavelength throughout the entire sweep. Thedetected photocurrent signal from the gas cell is digitized concurrentlywith the OCT interferogram and correlated with the known HF fingerprintto determine the wavenumber bias (k_(o)) of the swept source laser.Knowledge of wavenumber bias (k_(o)) allows accurate determination ofthe absolute wavenumber of each digitized sample throughout the spectralsweep, effectively removing any wavenumber offsets and/or phaseinstabilities in the laser source, wavemeter and sampling electronics.Knowledge of the magnitude of the fixed intervals and the opticalfrequency of at least one clock pulse provides knowledge of the opticalfrequency of every clock pulse provided by the optical clock.

In one configuration of an optical clock, an optical comb source may beused. An optical comb source provides light with a power spectraldensity that is uniformly spaced in the optical frequency domain at afixed, known, and stable optical frequency interval. By mixing orinterfering light emitted by the tunable laser source with light emittedby the optical clock, the interference signal provides an optical clockpulse. In this configuration, the instantaneous spectral linewidth ofthe tunable laser source is less than the optical frequency interval ofthe optical comb source. In addition, at least one of the clock pulsesgenerated by the optical clock is utilized to provide a referenceoptical frequency. For example, the first clock pulse generated by theoptical clock can correspond to an absorption line in a gas cell (e.g.,Hydrogen Fluoride). In this case the gas absorption line has a knownoptical frequency. Knowledge of the magnitude of the fixed intervals andthe optical frequency of at least one clock pulse provides knowledge ofthe optical frequency of every clock pulse provided by the opticalclock.

In another configuration of an optical clock, a Fabry-Perotinterferometer is used. In this configuration, light emitted by thetunable laser source is input into the Fabry-Perot interferometer. Thelight transmitted through the Fabry-Perot interferometer provides lightwith a power spectral density that is uniformly spaced in the opticalfrequency domain at a fixed optical frequency interval. In thisconfiguration, the instantaneous spectral linewidth of the tunable lasersource is less than the optical frequency interval of Fabry-Perotinterferometer. In addition, at least one of the clock pulses generatedby the optical clock is utilized to provide a reference opticalfrequency. For example, the first clock pulse generated by the opticalclock can correspond to an absorption line in a gas cell (e.g., HydrogenFluoride). In this case the gas absorption line has a known opticalfrequency. Knowledge of the magnitude of the fixed intervals and theoptical frequency of at least one clock pulse provides knowledge of theoptical frequency of every clock pulse provided by the optical clock.

In another embodiment, an enhanced detection of cancer with ultrasoundimaging 200 is provided. Ultrasonography is the ultrasound-baseddiagnostic imaging technique used to visualize muscles and internalorgans, their size, structures and any pathological lesions.“Ultrasound” applies to all acoustic energy with a frequency above humanhearing (20,000 Hertz or 20 kilohertz). Typical diagnostic sonographyscanners operate in the frequency range of 2 to 40 megahertz, hundredsof times greater than this limit. The choice of frequency is a trade-offbetween the image spatial resolution and penetration depth into thepatient, with lower frequencies giving less resolution and greaterimaging depth. Doppler ultrasonography uses the Doppler Effect to assesswhether blood is moving towards or away from a probe, and its relativevelocity. By calculating the frequency shift (υ_(D)) of a particularsample volume, for example a jet of blood flow over a heart valve, itsspeed and direction can be determined and visualized. Ultrasonagraphyand Doppler Ultrasonagraphy can best be understood by S. A. KanaIntroduction to physics in modern medicine, Taylor & Francis, (2003).The basic physics of the Doppler effect involving acoustic andelectromagnetic waves of OCT is similar and many of the signalprocessing techniques (hardware and software) used to estimate theDoppler shift of ultrasonic and optical coherence tomography signals isanalogous.

In one embodiment of the invention, an ultrasound probe 212 is coupledwith the magnetic field generator 100, as shown in FIG. 8. Ultrasound210 is directed into the body of the patient by known techniques and themoving red blood cells backscatter the ultrasonic energy back towardsthe transducer of the ultrasound. The oscillating magnetic fieldgenerated by the magnetic field generator 62 increases the contrast ofthe ultrasonic energy 210 received from the red blood cells. Thetransducer then converts the back-scattered ultrasonic energy 210 intoan electrical signal that is processed in some known manner to determinean estimate of the flow. An enhanced ultrasound image is produced, asdisplayed in FIG. 9.

In one example, a rectal ultrasound probe is coupled with a magnet toevaluate the prostate gland for cancer. Currently, ultrasound is usedfor prostate cancer screening; however, the approach provides poorsensitivity and specificity. Yet, all cancers are known in the art to behighly vascular, so then the application of the magnetic field bygenerator 62 coupled with ultrasound enhances the contrast availablefrom the endogenous RBC's in the prostate for cancer detection at anearlier stage. It is generally known in the art that tissues withcancerous cells have enhanced metabolic demand compared to normaltissues, so then cancerous cells have higher oxygen content fromhemoglobin and a greater concentration of deoxygenated hemoglobincompared to normal tissues. An exemplary ultrasound image for prostatecancer screening is shown in FIG. 9.

MM-ODT, OCT, and ultrasound coupled with an oscillating magnetic fieldall can be used in clinical management of patients who needmicrovasculature or vasculature monitoring, as shown in FIG. 10. Theimages could monitor and determine tissue perfusion and viabilitybefore, during and after therapeutic procedures. For example, the imagescould be used to detect oxygenated and deoxygenated blood supply, detectblood vessel location, spatial extent of cancer, vascular tissueabnormality identification, and imaging of port-wine stain.Alternatively, the images could monitor photodynamic therapy or evaluatecranial injuries. While medical applications of the invention have beendescribed, the embodiments of the invention are applicable to anycircumstance where image contrast is needed for fluids comprisingendogenous metallic compositions. MM-ODT, OCT, and ultrasound can beused in various combinations to detect vascular occurrences.

OCT Imaging Using Nanoparticles

Provided herein are methods, compositions and apparatuses for detectinga cell and/or a metallic composition using optical coherence tomography(“OCT”). By a “cell” is meant one or more cell of, or derived from, aliving organism or subject. The cell or cells can be located within asubject or can be located ex vivo. The disclosed methods, compositionsand apparatuses for detecting a cell and/or a metallic composition aredescribed herein variously by reference to cell(s), composition(s)and/or metallic composition(s). It will be understood that descriptionof various aspects of the disclosed methods, compositions andapparatuses by reference to one or a subset of cell(s), composition(s)or metallic composition(s) constitutes description of that aspect of thedisclosed methods, compositions and apparatuses to the non-referencedcell(s), composition(s) and metallic composition(s), unless the contextclearly indicates otherwise.

An exemplary method for detecting a cell comprises applying a magneticfield to the cell. A cell can comprise a cellular membrane and ametallic composition. Optionally, the metallic composition is a metallicnanoparticle that was administered to the subject or otherwise broughtinto contact with the cell. Optionally, the metallic composition ishemoglobin, as discussed previously.

The metallic composition can be located within the cell, including inthe cell's cellular membrane, or on the outside of the cell. If themetallic particle is located on the outside of the cell, it can beconnected or targeted to the exterior surface of the cell's cellularmembrane. If the metallic composition is located within the cell, it mayinclude inherent metallic compositions such as hemoglobin. Exemplarymethods of targeting or connecting a metallic composition to a cell aredescribed herein.

The applied magnetic field can interact with a metallic compositionlocated within the cell or located external and connected to the cell.The interaction of the magnetic field with the composition can cause achange in the cell. A change “in” the cell is not limited to changesinternal to the cell's cellular membrane. A change “in” the cell isinclusive of changes within the cell, and also includes any change to,of, or in the cell caused by the interaction of the magnetic field withthe composition. For example, changes that can occur “in” the cellinclude movement of the cell, movement of one or more cell components,movement of the metallic composition, a change in the cellular membranetension level of the cell, and a change in the internal strain field ofthe cell. Changes in the cell that cause changes, including those listedabove, of neighboring or surrounding cells or tissues can also bedetected. Thus, changes in a cell can cause changes in surrounding cellsor tissues. The changes in the surrounding cells or tissues can bedetected using the methods and systems described herein. Compositionslocated within or external to the cell can cause one or more detectablechanges in the cell when contacted by an applied magnetic field.

A detectable internal strain field can be generated in a cell when ametallic composition, including a metallic nanoparticle, is under theaction of an external force. The internal strain field can be detectedusing phase sensitive OCT using block correlation signal processingtechniques that have been applied in elasticity imaging in ultrasoundimaging. The external force may be provided by the application of anexternal magnetic flux density (B). Action of the external force on eachmetallic composition can produce movement of the metallic composition(z_(np)(t)) that produces a change in the cellular membrane tensionlevel or an internal strain field within a cell. Action of a force oneach metallic composition in a cell or tissues produces a movement ofthe metallic composition (z_(np)(t)). Movement of the metalliccomposition can be along the z-direction. The metallic composition canalso have movement in any direction which may be written as vectordisplacement, u_(np)(r_(o)) for a metallic composition positioned atr_(o). Metallic composition displacement u_(np)(r_(o)) can produce adisplacement field (u(r,r_(o))) in the proteins in the cell containingthe metallic composition and surrounding cells. In the case of ahomogeneous elastic media, the displacement field (u(r,r_(o))) can becomputed for a semi-infinite half-space following, for example, themethod of Mindlin (R. D. Mindlin, A force at a point of a semi-infinitesolid, Physics 1936, 7:195-202, which is incorporated by reference forthe methods taught therein). In the case of an inhomogeneousviscoelastic media, a finite element method numerical approach can beapplied to compute the displacement field in the cell. The displacementfield (u(r,r_(o))) produced by a metallic composition positioned atr_(o) can induce an internal stain field that is determined by change inthe displacement field along a particular direction. The strain field(∈_(ij)(r,r_(o))) is a tensor quantity and is given by,

${ɛ_{ij}\left( {r,r_{o}} \right)} = \frac{\partial{u_{i}\left( {r,r_{o}} \right)}}{\partial x_{j}}$

where u_(i)(r,r_(o)) is the i'th component of the displacement field andx_(j) is the j^(th) coordinate direction. For example, when j=3, x₃ isthe z-direction. The internal strain field in a cell due to all metalliccompositions in the cell and surrounding cells is a superposition of thestrain fields due to each metallic composition. A detectable change in acell can also be caused with light energy. For example, pulsed laserlight can be applied to contact a metallic particle comprised by thecell, where the metallic particle is included in a cell either naturallyoccurring or administered exogenously and the metallic particle isaffected by the pulsed laser light. The application of light energy cancause a detectable change in optical path due to a change in opticalrefractive and thermal elastic expansion. The light energy can alsocause motion of the cell, particle, or tissues proximate to the cell fordetection by optical coherence tomography or phase sensitive opticalcoherence tomography. Such movement can be caused by thermal elasticexpansion. Alternatively, sound energy can cause motion of the cell,particle, or tissues proximate to the cell for detection by opticalcoherence tomography or phase sensitive optical coherence tomography.

The change in the cell can be detected using optical coherencetomographic imaging modalities. Thus, the cell can be detected bydetecting the change in the cell caused by the interaction of themagnetic field with the metallic composition using such a modality. Thechange can be detected using a phase sensitive optical coherencetomographic imaging modality. Non-limiting examples of phase sensitiveoptical coherence tomographic (OCT) imaging modalities are describedherein. Phase sensitive OCT imaging modalities can comprise a probe fortransmitting and receiving light energy to and from the cell. The probecan be sized, shaped and otherwise configured for intravascularoperation. The probe can further comprise a magnetic source for applyingthe magnetic field to the cell. The magnetic field can be applied to thecell from a magnetic source located external to the subject or internalto the subject. The external source can be located in a probe or can bedistinct from a probe.

Metallic Nanoparticles

The metallic composition can comprise a plurality of metallicnanoparticles and/or a plurality of hemoglobin molecules. Thenanoparticles can be substantially spherical in shape and can have adiameter from about 0.1 nanometers (nm) to about 1000.0 nm. The size ofthe tetrametric hemoglobin protein is approximately 6 nm in diameter.The nanoparticles are not, however, limited to being spherical in shape.Thus, the nanoparticles are asymmetrical in shape. If the nanoparticlesare asymmetrical in shape, the largest cross sectional dimension of thenanoparticles can be from about 0.1 nanometers (nm) to about 1000.0 nmin length. The nanoparticles can be hemoglobin.

The metallic composition can comprise metal having non-zero magneticsusceptibility or zero magnetic susceptibility or combinations ofnon-zero and zero magnetic susceptibility metals. Thus, if thecomposition comprises nanoparticles, the nanoparticles can all have anon-zero magnetic susceptibility or a zero magnetic susceptibility or acombination of particles having a non-zero magnetic susceptibility and azero magnetic susceptibility. Metallic compositions having a non-zeromagnetic susceptibility can comprise a material selected from the groupconsisting of iron oxide, iron, cobalt, nickel, chromium andcombinations thereof. The metallic compositions can comprise metalhaving non-zero electrical conductivity or zero electrical conductivityor combinations of non-zero and zero electrical conductivity metals.Also provided is a method for detecting a composition, the methodwherein the composition comprises a magnetic or paramagnetic material.Any magnetic or paramagnetic material, whether metallic or non-metallic,can be used in the described methods or with the described systems. Inthis regard, any material can be used that can cause a change in a cellor can be detected using phase sensitive optical coherence tomographywhen contacted with an applied magnetic field. Similarly, non-metallic,not magnetic particles can be used to cause a change in a cell or can bedetected using phase sensitive optical coherence tomography whencontacted with an applied magnetic field using the methods and systemsdescribed herein.

The systems, apparatuses and methods can be practiced using metalliccompositions without magnetic susceptibility. When using metalliccompositions without magnetic susceptibility, or when using compoundshaving a non-zero magnetic susceptibility, an electrical eddy currentcan be induced in the composition.

To induce an eddy current in a metallic composition a first time-varyingmagnetic field can be applied to a cell. The first magnetic field caninteract with a metallic composition within or external to the cell toinduce an electrical eddy current within the metallic composition. Asecond magnetic field can be applied to the cell that interacts with theinduced eddy current to cause a change in the cell. The cell can bedetected by detecting the change in the cell caused by the interactionof the second magnetic field with eddy current using a phase sensitiveoptical coherence tomographic imaging modality. Exemplary changes in thecell caused by the interaction of the second magnetic field with theeddy current include movement of the cell, movement of the metalliccomposition, a change in the cellular membrane tension level, and achange in the internal strain field of the cell.

Thus, a metallic composition or a nanoparticle that does not have asignificant magnetic permeability can be used. For example, althoughgold nanoparticles do not have significant magnetic permeability manytarget-specific molecular agents (e.g., antibodies) can be conjugated tothe nanoparticle surface. When using a high-conductivity particle fordetection, a magnetic dipole can be induced in the particle by exposingto a time-varying magnetic field (B(t)).

The time-varying magnetic field (B(t)) can cause an electromotive forceor potential in the particle that can induce a volumetric and surfaceelectric eddy-current in the high-conductivity nanoparticle. Exemplarycircuitry for a magnetic pulser that can be used to produce an eddycurrent is described in G H Schroder, Fast pulsed magnet systems,Handbook of Accelerator Physics and Engineering, A. Chao and M. Tinger,Eds. 1998 or in IEEE transactions on instrumentation and measurement,VOL. 54, NO. 6, December 2005, pp 2481-2485, which are incorporatedherein by reference for the circuitry and methods described therein.

The eddy-current can produce time-varying magnetic moment that caninteract with a second applied magnetic field (B₂). The inducededdy-current in the high-conductivity nanoparticle or metalliccomposition and the second applied magnetic field can interact toproduce a torque or twist on the nanoparticle or metallic composition.The induced torque can twist the nanoparticle that is mechanicallylinked to a target in the cell (e.g., the membrane) or located insidethe cell. The twisting motion of the nanoparticle can modify theinternal strain field of the cell (surrounding cells and tissue) whichcan be detected using phase sensitive optical coherence tomography. Inthis approach, phase-sensitive data can be recorded before and afterapplication of a first field to induce an eddy current and blockcorrelation algorithms can be used to compute the depth resolved strainfield in the tissue resulting from the motion of the nanoparticle ormetallic composition.

In exemplary embodiments, large magnetic fields can be generated by lowtemperature superconducting magnets. These magnets need only be“charged” once, maintained at a low temperature and do not require anexternal current to maintain the magnetic field.

A metallic composition can be administered to the subject.Administration of exogenous metallic compositions, for example, metallicnanoparticles is described in greater detail below. Optionally, the cellcan be located within a subject and the metallic composition can beadministered to the subject. Optionally, the cell can be a macrophageand at least one metallic nanoparticle can be located within themacrophage or can be connected to the macrophage. The macrophage can belocated in an atherosclerotic plaque within the subject. The macrophagecan also be located within the eye of the subject.

The change in the cell caused by the interaction of the magnetic fieldwith the metallic composition can be detected by generating a phasesensitive optical coherence tomographic image. A phase sensitive opticalcoherence tomographic image can comprise one or more lines of phasesensitive light energy data captured using a phase sensitive opticalcoherence tomography modality, wherein at least one line is capturedduring the application of the magnetic field.

One or more data line can be produced by generating light energy andtransmitting at least a first portion of the generated light energy ontoa reference reflector wherein at least a portion of the transmittedfirst portion of light energy is reflected by the reference reflector.At least a second portion of the generated light energy can betransmitted to contact the cell wherein at least a portion of the lightenergy that contacts the cell is reflected by the cell. The light energyreflected by the reference reflector and by the cell can be received,and the received light energy can be combined, and the received lightenergy can interfere. The combined light energy is processed to producea phase sensitive optical coherence data line.

One or more data lines can also be produced by generating light energyand transmitting at least a first portion of the generated light energyonto a reference reflector wherein at least a portion of the transmittedfirst portion of light energy is reflected by the reference reflector.At least a second portion of the generated light energy can betransmitted to contact the metallic composition wherein at least aportion of the light energy that contacts the metallic composition isreflected by the composition. The light energy reflected by thereference reflector and by the composition can be received. The receivedlight energy can be combined, wherein the received light energyinterferes. The combined light energy can be processed to produce thephase sensitive optical coherence data line. One or more data lines canalso be produced by generating light energy and transmitting at least afirst portion of the generated light energy onto a reference reflectorwherein at least a portion of the transmitted first portion of lightenergy is reflected by the reference reflector. At least a secondportion of the generated light energy can be transmitted to contact themetallic composition wherein at least a portion of the light energy thatcontacts the metallic composition is reflected by the composition. Thelight energy reflected by the reference reflector and by the compositioncan be received. The received light energy can be combined, wherein thereceived light energy interferes. The combined light energy can beprocessed to produce the phase sensitive optical coherence data line.The phase sensitive optical coherence A-lines can be recorded before orafter application of the stimulating field (magnetic, eddy-current,generation of pulsed light energy). Thus, the methods can furthercomprise recording reference phase sensitive optical coherence A-lineprior to the non-lethal change and second phase sensitive opticalcoherence A-line during or after non-lethal change. The reference andsecond optical coherence A-line data can be correlated to quantify thenon-lethal change.

Phase sensitive light energy data lines can include the spectraldependent complex amplitude of light reflected from the cell, A_(c)(ν),where ν is the optical frequency of light. More precisely, what can bemeasured is product of the amplitudes of light reflected from the celland reference: A_(c)(ν)·A_(r)(ν)* where A_(r)(ν)* is the conjugate ofthe spectrally-dependent complex amplitude of light reflected from thereference. The quantity A_(c)(ν)·A_(r)(ν)* can be used to determineA_(c)(ν) the phase sensitive amplitude of light backreflected from thecell/tissue at different time-delays τ by using a time-frequencytransformation (e.g., Fourier).

A plurality of phase sensitive optical coherence A-lines can be capturedand used to construct an image. A phase sensitive image produced usingthe described systems and methods can have a phase sensitive resolutionof at least about 30.0 nanometers (nm), 25.0 nm, 15.0 nm, 10.0 nm, 5.0nm, 4.0 nm, 3.0 nm, or 2.0 nm. A plurality of phase sensitive opticalcoherence A-lines can be spatially and temporally distinct and the imagecan comprise a B-mode image frame of at least two of the data lines. Theplurality of phase sensitive light energy data lines can also betemporally distinct and the image can comprise an M-mode imagecomprising at least two of the lines.

When a plurality of lines are used to create an image, at least a firstphase sensitive light energy data line can be captured prior to theapplication of the magnetic field and at least a second phase sensitivelight energy data line can be captured during application of themagnetic field, or generally the external stimulus. The magnetic fieldstrength can be altered between the capture of data lines or between thecapture of images. For example, at least a first phase sensitive lightenergy data line can be captured during the application of the magneticfield, wherein the magnetic field has a first predetermined strength andat least a second phase sensitive light energy data line duringapplication of a second magnetic field having a second predeterminedstrength. The captured lines can be processed to create an image.Optionally, the first predetermined strength can be less than the secondpredetermined strength.

The described methods allows for the construction of both conventionalintensity based OCT B-scan images and phase sensitive B-scan images. Thephase sensitive B-scan images for viewing can correspond to changes inphase formed by at least two phase sensitive B-scan images correspondingto different magnetic field strengths (one of which can be zero magneticfield strength). At least two types of images can be viewed—one, aconventional intensity based OCT B-scan image and second a phasesensitive B-scan image formed by the difference of two phase sensitiveimages recorded at different magnetic field strengths.

Also provided are methods for detecting a composition comprising metalby applying a magnetic field to the composition, wherein the magneticfield interacts with the composition. The metallic composition can bedetected using a phase sensitive optical coherence tomographic imagingmodality. As described throughout, the composition can be located in acell or can be connected to a cell. The composition can also be locatedin connection with non-cellular biological matter. For example,non-cellular biological matter can include a protein, a lipid, apeptide, and a nucleic acid.

The methods of detecting cells and compositions using optical coherencetomography can comprise administering a plurality of metallicnanoparticles to a subject or imaging nanoparticles present within acell.

Optionally, at least one administered nanoparticle localizes within amacrophage located in the subject. At least one administerednanoparticle can also be optionally configured to localize to a targetsite in the subject. Optionally, at least one nanoparticle is presentwithin a cell. Optionally, the nanoparticle may be a hemoglobinmolecule.

In the methods described herein, a nanoparticle comprising a materialwith non-zero magnetic susceptibility can be positionally moved in vivoor in vitro by an applied magnetic field. A material of non-zeromagnetic susceptibility can include a variety of materials. For example,the nanoparticle can comprise any physiologically tolerable magneticmaterial or combinations thereof. The term magnetic material canoptionally include any material displaying ferromagnetic, paramagneticor superparamagnetic properties. For example, the nanoparticles cancomprise a material selected from the group consisting of iron oxide,iron, cobalt, nickel, and chromium. Metallic compositions as describedthroughout, including administered nanoparticles, can be magnetic.Optionally, a nanoparticle comprises iron oxide. When a nanoparticlecomprises metal or magnetic materials, it can be moved while in thesubject using an internally or externally applied magnetic field, asdescribed below. Any relevant metal with non-zero magneticsusceptibility or combinations thereof can be used. Many useable metalsare known in the art; however, any metal displaying the desiredcharacteristics can be used. Nanoparticles can also comprise acombination of a material with a non-zero magnetic susceptibility and amaterial with a lower or zero magnetic susceptibility. For example, goldcan be combined with higher magnetic susceptibility materials (e.g.,iron). For example, gold coated iron can be used. Nanoparticles can alsocomprise polymers or other coating materials alone or in combination.Such polymers or coating materials can be used to attach targetingligands, including but not limited to antibodies, as described below.When used in vivo, an administered nanoparticle can be physiologicallytolerated by the subject, which can be readily determined by one skilledin the art.

Nanoparticles can be solid, hollow or partially hollow and can bespherical or asymmetrical in shape. Optionally, the cross section of anasymmetric nanoparticle is oval or elliptical. As one of skill in theart will appreciate, however, other asymmetric shapes can be used. Thenanoparticles can comprise shelled or multi-shelled nanoparticles.Shelled or multi-shelled nanoparticles can have targeting ligandsconjugated to the shell material wherein the targeting ligand has anaffinity for or binds to a target site in a subject or ex vivo. Suchshelled or multi-shelled nanoparticles can be made, for example, usingtechniques known in the art, for example, as described in Loo et al.,“Nanoshell-Enabled Photonics-Based Imaging and Therapy of Cancer,” Tech.Cancer Res. and Treatment, (2004) 3(1) 33-40, which is incorporatedherein by reference for the methods taught herein. Further, Oldenburg etal., “Nanoengineering of Optical Resonances,” Chemical Physics Letters(1998) 288, 243-247, is incorporated herein for methods of nanoshellsynthesis.

Localizing Nanoparticles

A metallic composition, including a nanoparticle, can be configured tolocalize to a target site within the subject. For example, the metalliccomposition can be configured to localize to a neoplastic cell, to apeptide, to a protein, or to a nucleic acid. Optionally, the target siteis an extracellular domain of a protein. A variety of cell types canalso be targets of the metallic compositions. For example, target cellscan be selected from one or more of a neoplastic cell, a squameous cell,a transitional cell, a basal cell, a muscle cell, an epithelial cell,and a mucosal cell. The target cells can also be located at differentanatomical locations within a subject. For example, the cell can belocated in the subject at an anatomical location selected from the groupconsisting of a lung, bronchus, intestine, stomach, colon, eye, heart,blood vessel, cervix, bladder, urethra, skin, muscle, liver, kidney, andblood.

One or more administered nanoparticle can localize to a desired targetwithin the subject using passive or active targeting mechanisms. Passivetargeting mechanisms take advantage of the subject's inherent defensemechanisms to highlight phagocytic cells naturally responsible forparticle clearance. For example, macrophage rich areas are apathological correlate to an unstable atherosclerotic plaque in asubject. Moreover, administered nanoparticles, for example, smallsuperparamagneitc and ultrasmall superparamagnetic particles of ironoxide, are avidly taken up, or engulfed by, macrophages located inunstable plaques. Thus, through the subject's natural defense mechanism,wherein macrophages accumulate in an unstable atherosclerotic plaque andengulf administered nanoparticles, administered nanoparticles canpassively target the unstable plaque. Similarly, macrophages located inthe eye of a subject can engulf nanoparticles. Such passive targeting ofnanoparticles can be used with the methods and apparatuses describedherein to highlight a plaque's instability or to highlight otheraccumulation of phagocytic cells.

Active targeting mechanisms can refer to the use of ligand-directed,site-specific targeting of nanoparticles. A nanoparticle can beconfigured to localize to a desired target site in a subject using awide variety of targeting ligands including, but not limited to,antibodies, polypeptides, peptides, nucleic acids, and polysaccharides.Such nanoparticles are referred to herein as “targeted nanoparticles.”Targeting ligands or fragments thereof can be used to target ananoparticle to cellular, or other endogenous or exogenous biomarkers inthe subject. Such a biomarkers or “target sites” can include, but arenot limited to, proteins, polypeptides, peptides, polysaccharides,lipids, or antigenic portions thereof, which are expressed within thesubject. When active targeting mechanisms are used to target a cell, thetargeted nanoparticle can be optionally internalized by the targetedcell.

Thus, using the disclosed methods, at least one administerednanoparticle can optionally localize within a macrophage located in thesubject and/or at least one administered targeted nanoparticle canlocalize to a desired target site in the subject.

The methods and apparatuses are not, however, limited to in vivoadministration to a subject. As would be clear to one skilled in theart, nanoparticles, including targeted nanoparticles, can beadministered in vitro to an ex vivo sample with localization of thenanoparticle to a desired target site and subsequent imaging occurringin vitro. Moreover, a composition, including at least one nanoparticlecan be administered to a subject in vivo, and a sample can besubsequently taken from the subject and imaged ex vivo using themethods, systems, and apparatuses described herein.

When using a targeted nanoparticle the target site in vivo or in vitrocan be endogenous or exogenous. The target site can be selected from thegroup consisting of an organ, cell, cell type, blood vessel, thrombus,fibrin and infective agent antigens or portions thereof. Optionally, thetarget site can be a neoplastic cell. The target site can also be anextracellular domain of a protein. Furthermore, the target site can beselected from the group consisting of a lung, bronchus, intestine,stomach, colon, heart, brain, blood vessel, cervix, bladder, urethra,skin, muscle, liver, kidney and blood. The target site can also be acell. For example, a cell can be selected from the group consisting of,but not limited to, a neoplastic cell, a squameous cell, a transitionalcell, a basal cell, a muscle cell, an epithelial cell, a lymphocyte, aleukocyte, a monocyte, a red blood cell, and a mucosal cell.

Thus, targeted nanoparticles can be targeted to a variety of cells, celltypes, antigens (endogenous and exogenous), epitopes, cellular membraneproteins, organs, markers, tumor markers, angiogenesis markers, bloodvessels, thrombus, fibrin, and infective agents. For example, targetednanoparticles can be produced that localize to targets expressed in asubject. Optionally, the target can be a protein, and can be a proteinwith an extracellular or transmembrane domain. Optionally, the targetcan be the extracellular domain of a protein.

Desired targets can be based on, but not limited to, the molecularsignature of various pathologies, organs and/or cells. For example,adhesion molecules such as integrin αvβ3, intercellular adhesionmolecule-1 (I-CAM-1), fibrinogen receptor GPIIb/IIIa and VEGF receptorsare expressed in regions of angiogenesis, inflammation or thrombus.These molecular signatures can be used to localize nanoparticles throughthe use of a targeting ligand. The methods described herein optionallyuse nanoparticles targeted to one or more of VEGFR2, I-CAM-1, αvβ3integrin, αv integrin, fibrinogen receptor GPIIb/IIIa, P-selectin,and/or mucosal vascular adressin cell adhesion molecule-1.

As used in this invention, the term “epitope” is meant to include anydeterminant capable of specific interaction with a targeting ligand asdescribed below. Epitopic determinants can consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand can have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Targeting ligands specific for a molecule that is expressed orover-expressed in a cell, tissue, or organ targeted for imaging, such aspre-cancerous, cancerous, neoplastic, or hyperproliferative cells,tissues, or organs, can be used with the nanoparticles described herein.This use can include the in vivo or in vitro imaging, detection, ordiagnosis of pre-cancerous, cancerous, neoplastic or hyperproliferativecells in a tissue or organ. The compositions and methods of theinvention can be used or provided in diagnostic kits for use indetecting and diagnosing cancer.

As used herein, a targeted cancer to be imaged, detected or diagnosedcan be selected from, but are not limited to, the group comprisinglymphomas (Hodgkins and non-Hodgkins), B cell lymphoma, T cell lymphoma,myeloid leukemia, leukemias, mycosis fungoides, carcinomas, carcinomasof solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas,gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas,melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas orsarcomas, metastatic cancers, bladder cancer, brain cancer, nervoussystem cancer, squamous cell carcinoma of head and neck,neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer,melanoma, squamous cell carcinomas of the mouth, throat, larynx, andlung, colon cancer, cervical cancer, cervical carcinoma, breast cancer,epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer,esophageal carcinoma, head and neck carcinoma, hematopoietic cancers,testicular cancer, colo-rectal cancers, prostatic cancer, or pancreaticcancer.

Pre-cancerous conditions to be imaged, detected or diagnosed include,but are not limited to, cervical and anal dysplasias, other dysplasias,severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.As would be clear to one skilled in the art, however, additional cancersand pre-cancerous conditions can be imaged, detected or diagnosed usingthe methods and apparatuses described herein.

Using methods known in the art, and as described herein, targetingligands, such as polyclonal or monoclonal antibodies, can be produced todesired target sites in a subject. Thus, a targeted nanoparticle canfurther comprise an antibody or a fragment thereof. Methods forpreparing and characterizing antibodies are well known in the art (See,e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988; incorporated herein by reference for the methods taught therein).

Monoclonal antibodies can be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that can be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies of the invention can be madeusing the hybridoma method first described by Kohler & Milstein, Nature256:495 (1975), or can be made by recombinant DNA methods (Cabilly, etal., U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster can be immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theantigen used for immunization. Alternatively, lymphocytes can beimmunized in vitro. Lymphocytes can be then fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

DNA encoding a monoclonal antibody can be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells can serve as apreferred source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which can then be transfected into host cells suchas simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci.81, 6851 (1984), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies can beprepared that have the binding specificity of an anti-cancer,pre-cancer, or hyperproliferative cell or other target molecule.Optionally, the antibody used herein is “humanized” or fully human.

Non-immunoglobulin polypeptides can be substituted for the constantdomains of an antibody of the invention, or they can be substituted forthe variable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody comprising oneantigen-combining site having specificity for a first antigen andanother antigen-combining site having specificity for a differentantigen.

Chimeric or hybrid antibodies also can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332,323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies, wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

Antibodies can be humanized with retention of high affinity for thetarget site antigen and other favorable biological properties. Humanizedantibodies can be prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e. theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget site antigen(s), can be achieved. In general, the CDR residuesare directly and most substantially involved in influencing antigenbinding.

Human monoclonal antibodies can be made by a hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987).

Transgenic animals (e.g., mice) can be used that are capable, uponimmunization, of producing a repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy chainjoining region (JH) gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90,2551-255 (1993); Jakobovits et al., Nature 362, 255-258 (1993).

Alternatively, phage display technology (McCafferty et al., Nature 348,552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced can confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 (1992)). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This technique allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993),and the isolation of a high affinity human antibody directly from suchlarge phage library is reported by Griffith et al., EMBO J. (1994). Geneshuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting,” the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published Apr. 1, 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. One of the binding specificities is for a first antigen andthe other one is for a second antigen.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al., EMBO10, 3655-3659 (1991). For further details ofgenerating bispecific antibodies see, for example, Suresh et al.,Methods in Enzymology 121, 210 (1986).

Heteroconjugate antibodies are also within the scope of the describedcompositions and methods. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Heteroconjugate antibodies can be madeusing any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

A variety of immunoassay formats can be used to select antibodies thatselectively bind with a desired target site or target site antigen. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a protein, protein variant,or fragment thereof. See Harlow and Lane. Antibodies, A LaboratoryManual. Cold Spring Harbor Publications, New York, (1988), for adescription of immunoassay formats and conditions that could be used todetermine selective binding. The binding affinity of a monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

Not only can a targeted nanoparticle comprise an antibody or fragmentthereof, but a targeted nanoparticle can also comprise targeting ligandthat is a polypeptide or a fragment thereof. Optionally, polypeptidesthat are internalized by target cells can be attached to the surface ofa nanoparticle. Ligands that are internalized can optionally be used forinternalization of a nanoparticle into a target cell. A modified phagelibrary can be use to screen for specific polypeptide sequences that areinternalized by desired target cells. For example, using the methodsdescribed in Kelly et al., “Detection of Vascular Adhesion Molecule-1Expression Using a Novel Multimodal Nanoparticle,” Circulation Res.,(2005) 96:327-336, which is incorporated herein for the methods taughttherein, polypeptides can be selected that are internalized by VCAM-1expressing cells or other cells expressing a ligand of interest.

There are a number of methods for isolating proteins which can bind adesired target. For example, phage display libraries have been used toisolate numerous polypeptides that interact with a specific target. (Seefor example, U.S. Pat. Nos. 6,031,071; 5,824,520; 5,596,079; and5,565,332 which are herein incorporated by reference at least for theirmaterial related to phage display and methods related to combinatorialchemistry). Thus targeted nanoparticles can comprise a polypeptide orfragments thereof that interact with a desired target. A targetednanoparticle can also comprise a binding domain of an antibody or phage.

The term “polypeptide” or “peptide” is used broadly herein to mean twoor more amino acids linked by a peptide bond. The term “fragment” or“proteolytic fragment” also is used herein to refer to a product thatcan be produced by a proteolytic reaction on a polypeptide, i.e., apeptide produced upon cleavage of a peptide bond in the polypeptide. Afragment can be produced by a proteolytic reaction, but it should berecognized that a fragment need not necessarily be produced by aproteolytic reaction but can be produced using methods of chemicalsynthesis or methods of recombinant DNA technology, to produce asynthetic peptide that is equivalent to a proteolytic fragment. Itshould be recognized that the term “polypeptide” is not used herein tosuggest a particular size or number of amino acids comprising themolecule, and that a polypeptide of the invention can contain up toseveral amino acid residues or more.

A nanoparticle can bind selectively or specifically to a desired targetsite, and/or can be internalized by a target cell. Such selective orspecific binding and/or internalization can be readily determined usingthe methods, systems and apparatuses described herein. For example,selective or specific binding can be determined in vivo or in vitro byadministering a targeted nanoparticle and detecting an increase in lightscattering from the nanoparticle bound to a desired target site orinternalized into the desired target cell. Detection of light scatteringcan be measured using the systems and apparatuses described below.

Thus, a targeted nanoparticle can be compared to a control nanoparticlehaving all the components of the targeted nanoparticle except thetargeting characteristics, such as, for example, targeting ligand. Bydetecting phase sensitive image data from the targeted nanoparticlebound to a desired target site versus a control nanoparticle, thespecificity or selectivity of binding or internalization can bedetermined. If an antibody, polypeptide, or fragment thereof, or othertargeting ligand is used, selective or specific binding to a target canbe determined based on standard antigen/polypeptide/epitope/antibodycomplementary binding relationships. Further, other controls can beused. For example, the specific or selective targeting of thenanoparticles can be determined by exposing targeted nanoparticles to acontrol tissue, which includes all the components of the test or subjecttissue except for the desired target ligand or epitope. To compare acontrol sample to a test sample, levels of light scattering can bedetected by, for example, the systems described below and the differencein levels or location can be compared.

A targeting ligand can be coupled to the surface or shell of at leastone of the nanoparticle. Targeted nanoparticles comprising targetingligands can be produced by methods known in the art. For exampleligands, including but not limited to, antibodies, peptides,polypeptides, or fragments thereof can be conjugated to the nanoparticlesurface.

Any method known in the art for conjugating a targeting ligand to ananoparticle can be employed, including, for example, those methodsdescribed by Hunter, et al., Nature 144:945 (1962); David, et al.,Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).Established protocols have been developed for the labeling metallicnanoparticles with a broad range of biomolecules, including protein A,avidin, streptavidin, glucose oxidase, horseradish peroxidase, and IgG(antibodies). Nanoparticles can be prepared with bioorganic molecules ontheir surface (DNA, antibodies, avidin, phospholipids, etc). Thenanoparticles can be characterized, modified, and conjugated withorganic and biomolecules. Polymers or other intermediate molecules canbe used to tether antibodies or other targeting ligands to the surfaceof nanoparticles. Methods of tethering ligands to nanoparticles are knowin the art as described in, for example, Loo et al., “Nanoshell-EnabledPhotonics-Based Imaging and Therapy of Cancer,” Tech. Cancer Res. andTreatment, (2004) 3(1) 33-40, which is incorporated herein by referencefor the methods taught herein.

Covalent binding of a targeting ligand to a nanoparticle can beachieved, for example, by direct condensation of existing side chains orby the incorporation of external bridging molecules. Many bivalent orpolyvalent agents can be useful in coupling polypeptide molecules toother particles, nanoparticles, proteins, peptides or amine functions.Examples of coupling agents are carbodiimides, diisocyanates,glutaraldehyde, diazobenzenes, and hexamethylene diamines. This list isnot intended to be exhaustive of the various coupling agents known inthe art but, rather, is exemplary of the more common coupling agentsthat can be used.

Optionally, one can first derivatize an antibody if used, and thenattach the nanoparticle to the derivatized product. As used herein, theterm “derivatize” is used to describe the chemical modification of theantibody substrate with a suitable cross-linking agent. Examples ofcross-linking agents for use in this manner include the disulfide-bondcontaining linkers SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate)and SMPT(4-succinimidyl-oxycarbonyl-α-methyl-α(2-pyridyldithio)toluene).

Targeting ligands can also be conjugated to nanoparticles using methodsincluding the preparation of biotinylated antibody molecules and theirconsequent interaction with streptavidin/nanoparticle conjugates. Thisapproach takes advantage of strong biospecific interaction betweenbiotin and streptavidin and known protocols for immobilization ofstreptavidin on nanoparticles. Polypeptides with thiol terminated alkylchains can be directly attached to the surface of nanoparticles usingthe procedures described in Elghanian, R., et al., Selectivecolorimetric detection of polynucleotides based on thedistance-dependent optical properties of gold nanoparticles. Science,1997. 277 (5329): p. 1078-1080 (incorporated by reference for themethods taught therein). For conjugation procedure one can use a mixtureof thiol terminated polypeptides and relatively small mercaptoaceticmolecules to avoid high density immobilization of the polypeptides.

Targeted nanoparticles can be prepared with a biotinylated surface andan avidinated antibody, peptide, polypeptide or fragment thereof can beattached to the nanoparticle surface using avidin-biotin bridgingchemistry. Avidinated nanoparticles can be used and a biotinylatedantibody or fragment thereof or another biotinylated targeting ligand orfragments thereof can be administered to a subject. For example, abiotinylated targeting ligand such as an antibody, protein or otherbioconjugate can be used. Thus, a biotinylated antibody, targetingligand or molecule, or fragment thereof can bind to a desired targetwithin a subject. Once bound to the desired target, the nanoparticlewith an avidinated surface can bind to the biotinylated antibody,targeting molecule, or fragment thereof. When bound in this way, lightenergy can be transmitted to the bound nanoparticle, which can producelight scattering of the transmitted light. An avidinated nanoparticlecan also be bound to a biotinylated antibody, targeting ligand ormolecule, or fragment thereof prior to administration to the subject.

When using a targeted nanoparticle with a biotinylated surface or anavidinated surface a targeting ligand can be administered to thesubject. For example, a biotinylated targeting ligand such as anantibody, polypeptide or other bioconjugate, or fragment thereof, can beadministered to a subject and allowed to accumulate at a target site

When a targeted nanoparticle with a biotinylated surface is used, anavidin linker molecule, which attaches to the biotinylated targetingligand can be administered to the subject. Then, a targeted nanoparticlewith a biotinylated shell can be administered to the subject. Thetargeted nanoparticle binds to the avidin linker molecule, which isbound to the biotinylated targeting ligand, which is itself bound to thedesired target. In this way, a three step method can be used to targetnanoparticles to a desired target. The targeting ligand can bind to allof the desired targets detailed above as would be clear to one skilledin the art.

Nanoparticles, including targeted nanoparticles, can also comprise avariety of markers, detectable moieties, or labels. Thus, for example, ananoparticle equipped with a targeting ligand attached to its surfacecan also include another detectable moiety or label. As used herein, theterm “detectable moiety” is intended to mean any suitable label,including, but not limited to, enzymes, fluorophores, biotin,chromophores, radioisotopes, colored particles, electrochemical,chemical-modifying or chemiluminescent moieties. Common fluorescentmoieties include fluorescein, cyanine dyes, coumarins, phycoerytluin,phycobiliproteins, dansyl chloride, Texas Red, and lanthanide complexes.Of course, the derivatives of these compounds are included as commonfluorescent moieties.

The detection of the detectable moiety can be direct provided that thedetectable moiety is itself detectable, such as, for example, in thecase of fluorophores. Alternatively, the detection of the detectablemoiety can be indirect. In the latter case, a second moiety reactablewith the detectable moiety, itself being directly detectable can beemployed.

A composition, including at least one nanoparticle, can be administeredto a subject orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like. Parenteral administration of acomposition, if used, is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution ofsuspension in liquid prior to injection, or as emulsions.

The compositions, including nanoparticles, can be used in combinationwith a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material can be administered to a subject, alongwith the nanoparticle, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5.0 to about 8.0, and more preferably fromabout 7.0 to about 7.5. As described above, compositions can beadministered intravascularly. Administered compositions can includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the composition of choice.Administered compositions can also include one or more activeingredients such as antimicrobial agents, anti-inflammatory agents,anesthetics, and the like.

When used in the described methods, an effective amount of one of thecompositions, including the nanoparticles, of the present invention canbe determined by one skilled in the art. The specific effective doselevel for any particular subject can depend upon a variety of factorsincluding the type and location of the target site, activity of thespecific composition employed, the specific composition employed, theage, body weight, general health, sex and diet of the subject, the timeof administration, the route of administration, the rate of excretion ofthe specific composition employed, the duration of the treatment, drugsused in combination or coincidental with the specific compositionemployed, and like factors well known in the medical arts. For example,it is well within the skill of the art to start doses of the compositionat levels lower than those required to achieve the desired diagnostic orimaging effect and to gradually increase the dosage until the desiredeffect is achieved. If desired, an effective dose can be divided intomultiple doses for purposes of administration.

Depending on the exemplary factors above, on the composition used, onthe intended target site for the composition, and whether active orpassive targeting of the described compositions is used, the timebetween administration of the described compositions and the detectionof the described nanoparticles within the subject can vary. For example,detection of the described nanoparticles can be performed at one or moretime seconds, minutes, hours, days, and/or weeks after administration ofthe compositions to the subject. When and how frequently methods ofdetection of an administered composition are performed can be determinedby one skilled in the art through routine administration and detection.

OCT System

Also provided herein are systems for detecting a cell or metalliccomposition. An exemplary system comprises a magnet for applying amagnetic field to a cell and a phase sensitive optical coherencetomographic imaging modality for detecting the cell and/or metalliccomposition. The phase sensitive optical coherence tomographic imagingmodality can comprise a probe for transmitting and receiving lightenergy to and from the cell. The probe can be an intravascular probe.

The phase sensitive optical coherence tomographic imaging modalityincluded in the system can comprise a light source, a light splitter, aprobe and a reference reflector. The phase sensitive optical coherencetomographic imaging modality may include the light splitter, but doesnot need to include a light splitter, i.e. a light coupler and the likemay be used. Light energy generated by the light source can betransmitted to and split by the splitter for transmission to thereference reflector and to the probe. The probe can be configured totransmit at least a portion of the light energy transmitted thereto intoa target cell and to receive reflected light energy from the target celland the reference reflector can be configured to reflect at least aportion of the light energy transmitted thereto. The system can furtherinclude a processor for processing reflected light energy from thereference reflector and light energy received by the probe to produce aphase sensitive optical coherence A-line. The reference reflector can belocated in the probe.

Although the exemplary systems described also include a multitude offibers in the sample path, the described systems and methods are notintended to be limited to embodiments having a multitude of fibers.Thus, systems comprising one fiber and methods of using such exemplarysystems are covered. Optionally, an exemplary system comprises a probehaving a single optical fiber and a rotary reflector in opticalcommunication with the single optical fiber.

FIG. 6 is a block diagram illustrating an exemplary system 100 that canbe used for performing the imaging methods with nanoparticles. Thenanoparticles are imaged in the blood flow or in the surrounding bloodvessels of the blood flow. These exemplary OCT systems are only examplesof phase sensitive spectral domain OCT systems and are not intended tosuggest any limitation as to the scope of use or functionality of OCTarchitectures. Neither should the OCT systems be interpreted as havingany dependency nor a requirement relating to any one or combination ofcomponents illustrated in the exemplary OCT systems.

The OCT system 100 of FIG. 6 includes a general-purpose computing devicein the form of a computer 101. The components of the computer 101 caninclude, but are not limited to, one or more processors or processingunits 103, a system memory 112, and a system bus 113 that couplesvarious system components including the processor 103 to the systemmemory 112.

The system bus 113 represents one or more of several possible types ofbus structures, including a memory bus or memory controller, aperipheral bus, an accelerated graphics port, and a processor or localbus using any of a variety of bus architectures. By way of example, sucharchitectures can include an Industry Standard Architecture (ISA) bus, aMicro Channel Architecture (MCA) bus, an Enhanced ISA (EISA) bus, aVideo Electronics Standards Association (VESA) local bus, and aPeripheral Component Interconnects (PCI) bus also known as a Mezzaninebus. This bus, and all buses specified in this description can also beimplemented over a wired or wireless network connection. The bus 113,and all buses specified in this description can also be implemented overa wired or wireless network connection and each of the subsystems,including the processor 103, a mass storage device 104, an operatingsystem 105, an image construction software 106, a nanoparticle movementimage construction software 107, light signal data 108, the systemmemory 112, an OCT input interface 111, an OCT output interface 110, adisplay adapter 109, a display device 127, a human interface device 102,and a digital image capture device 117, can be contained within one ormore remote computers (not shown) at physically separate locations,connected through buses of this form, in effect implementing a fullydistributed system.

The computer 101 can include a variety of computer readable media. Suchmedia can be any available media that is accessible by the computer 101and includes both volatile and non-volatile media, removable andnon-removable media.

The system memory 112 includes computer readable media in the form ofvolatile memory, such as random access memory (RAM), and/or non-volatilememory, such as read only memory (ROM). The system memory 112 typicallycontains data such as light signal data 108 and/or program modules suchas operating system 105, image construction software 106 andnanoparticle movement (or cellular membrane tension level or internalstrain field change) image construction software 107 that areimmediately accessible to and/or are presently operated on by theprocessing unit 103. Throughout this application the disclosed methods,compositions and apparatuses for detecting a cell and/or a metalliccomposition are described herein variously by reference to metallicparticle movement, cellular movement, changes in cellular tension level,changes in internal strain field of a cell, and change in neighboring orsurrounding cells and/or tissues(s). It will be understood thatdescription of various aspects of the disclosed methods, compositionsand apparatuses by reference to detecting one or more of metallicparticle movement, cellular movement, changes in cellular tension level,changes in internal strain field of a cell, and change in neighboring orsurrounding cells and/or tissues(s) constitutes description of thataspect of the disclosed methods, compositions and apparatuses to thenon-referenced detection of metallic particle movement, cellularmovement, changes in cellular tension level, changes in internal strainfield of a cell, and change in neighboring or surrounding cells and/ortissues(s), unless the context clearly indicates otherwise. Thus,nanoparticle movement image construction software can also include oralternatively include cellular movement, changes in cellular tensionlevel, changes in internal strain field of a cell, and change inneighboring or surrounding cells and/or tissues(s) image constructionsoftware.

The computer 101 can also include other removable/non-removable,volatile/non-volatile computer storage media. By way of example, FIG. 6illustrates a mass storage device 104 which can provide non-volatilestorage of computer code, computer readable instructions, datastructures, program modules, and other data for the computer 101. Forexample, a mass storage device 104 can be a hard disk, a removablemagnetic disk, a removable optical disk, magnetic cassettes or othermagnetic storage devices, flash memory cards, CD-ROM, digital versatiledisks (DVD) or other optical storage, random access memories (RAM), readonly memories (ROM), electrically erasable programmable read-only memory(EEPROM), and the like.

Any number of program modules can be stored on the mass storage device104, including by way of example, an operating system 105, imageconstruction software 106, nanoparticle movement (or cellular membranetension level or internal strain field change) image constructionsoftware 107, and light signal data 108. Each of the operating system105, image construction software 106, nanoparticle movement (or cellularmembrane tension level or internal strain field change) imageconstruction software 107, light signal data 108 (or some combinationthereof) can include elements of the programming image constructionsoftware 106 and the nanoparticle movement (or cellular membrane tensionlevel or internal strain field change) image construction software 107.

A user can enter commands and information into the computer 101 via aninput device (not shown). Examples of such input devices include, butare not limited to, a keyboard, pointing device (e.g., a “mouse”), amicrophone, a joystick, a serial port, a scanner, and the like. Theseand other input devices can be connected to the processing unit 103 viaa human machine interface 102 that is coupled to the system bus 113, butcan be connected by other interface and bus structures, such as aparallel port, game port, or a universal serial bus (USB).

A display device 127 can also be connected to the system bus 113 via aninterface, such as a display adapter 109. For example, a display devicecan be a monitor. In addition to the display device 127, other outputperipheral devices can include components such as speakers (not shown)and a printer (not shown) which can be connected to the computer 101 viaan input/output interface (not shown).

The computer 101 can operate in a networked environment using logicalconnections to one or more remote computing devices (not shown). By wayof example, a remote computing device can be a personal computer,portable computer, a server, a router, a network computer, a peer deviceor other common network node, and so on.

Logical connections between the computer 101 and a remote computingdevice (not shown) can be made via a local area network (LAN) and ageneral wide area network (WAN). Such networking environments arecommonplace in offices, enterprise-wide computer networks, intranets,and the Internet. In a networked environment, image constructionsoftware 106, nanoparticle movement (or cellular membrane tension levelor internal strain field change) image construction software 107 andlight signal data 108 depicted relative to the computer 101, or portionsthereof, can be stored in a remote memory storage device (not shown).For purposes of illustration, application programs and other executableprogram components such as the operating system are illustrated hereinas discrete blocks, although it is recognized that such programs andcomponents reside at various times in different storage components ofthe computing device 101, and are executed by the data processor(s) ofthe computer.

An implementation of the image construction software 106 and thenanoparticle movement (or cellular membrane tension level or internalstrain field change) image construction software 107 can be stored on ortransmitted across some form of computer readable media. Computerreadable media can be any available media that can be accessed by acomputer. By way of example, and not limitation, computer readable mediacan comprise “computer storage media” and “communications media.”“Computer storage media” include volatile and non-volatile, removableand non-removable media implemented in any method or technology forstorage of information such as computer readable instructions, datastructures, program modules, or other data. Computer storage mediaincludes, but is not limited to, RAM, ROM, EEPROM, flash memory or othermemory technology, CD-ROM, digital versatile disks (DVD) or otheroptical storage, magnetic cassettes, magnetic tape, magnetic diskstorage or other magnetic storage devices, or any other medium which canbe used to store the desired information and which can be accessed by acomputer.

The light signal data 108 can enter the computer 101 via the OCT inputinterface 111. The OCT output interface can be IEEE-488, IEEE-1394,Universal Serial Bus (USB), or the like. The light signal data 108 canbe stored in mass storage device 104 and transferred to system memory112 as light signal data 108 to be used by image construction software106 and nanoparticle movement (or cellular membrane tension level orinternal strain field change) image construction software 107.

The OCT output interface 110 connects the computer 101 to a magnetcontrol 114. This connection can allow a user to regulate the currentsent to a magnet 115 and a magnet 116 by the magnet control 114. Themagnet control 114 directs current flow into the magnets 115 or 116. Themagnet control 114 can work in conjunction with a line scan camera 139so that a user-specified field pulse sequence is present at the scanningsite.

FIG. 6 illustrates an example of a Phase Sensitive OCT system 100. ThePhase Sensitive OCT system 100 can be utilized in conjunction with thecomputer and network architectures described above.

The Phase Sensitive OCT system 100 can include a general-purposecomputing device in the form of a computer 101 and all subsystems of thecomputer 101, as previously described. The Phase Sensitive OCT system100 can also include, as previously described, a display device 127, amagnet control 114, and a magnet 116.

Light energy can be generated by a light source 117. The light source117 can be a broadband laser light source coupled into optical fiberemitting light energy over a broad range of optical frequencies. Forexample, the range can be from about 400 nanometers to about 1600nanometers. The light energy can be emitted over a multiplicity ofoptical wavelengths or frequencies. Alternatively, the light source canbe a narrowband tunable laser light source wherein the opticalwavelengths generated range from about 400 nanometers to about 1600nanometers. The light spectrum is continually varied in time, over aspecified spectral region. As used herein, optical fiber can refer toglass or plastic wire or fiber. Optical fiber is indicated on FIG. 6 aslines connecting the various blocks of the figures. Where light energyis described as “passing,” “traveling,” “returning,” “directed,” orsimilar movement, such movement can be via optical fiber.

A fraction of the generated light energy passes from the light source117 into an optical spectrum analyzer 118. The optical spectrum analyzer118 measures optical frequency as the light energy is emitted from thelight source 117 as a function of time. The optical spectrum analyzer118 samples a portion of the light emitted by the light source 117. Theoptical spectrum analyzer 118 monitors the power spectral density oflight entering the splitter 119. The remaining fraction of light energyfrom the light source 117 passes into a splitter 119. The splitter 119can be a device with four ports. Port 1 allows light energy to enter thesplitter 119. Ports 2 and 3 allow light energy to leave and re-enter thesplitter 119. Port 4 allows light energy to leave the splitter 119. Thesplitter 119 couples the light into Port 1. The splitter 119 divides thelight according to a pre-determined split ratio selected by a user. Forexample, the split ratio can be 50/50 wherein half of the light energyentering the splitter 119 at Port 1 exits the splitter 119 through Port2 and half exits the splitter 119 through Port 3. In another nonlimiting example, the split ratio can be 60/40 wherein 60% of the lightenergy passes through Port 2 and 40% of the light energy passes throughPort 3.

A fraction of the light energy (determined by the split ratio) thatexits the splitter 119 through Port 2 travels to a reference reflectorsurface 120. The light energy is reflected from the reference reflectorsurface 120 back to the splitter 119 into Port 2. The referencereflector can be, by way of example, but not limitation, a planarmetallic mirror or a multilayer dielectric reflector with a specifiedspectral amplitude/phase reflectivity. The remaining fraction of lightthat entered splitter 119 through Port 1 exits splitter 119 through Port3 and enters an OCT probe 122. The OCT probe 122 can be a turbine-typecatheter as described in Patent Cooperation Treaty applicationPCT/US04/12773 filed Apr. 23, 2004 which claims priority to U.S.provisional application 60/466,215 filed Apr. 28, 2003, each hereinincorporated by reference for the methods, apparatuses and systemstaught therein. The OCT probe 122 can be located within a subject 121 toallow light reflection off of subject 121 tissues and nanoparticles 123.

The light energy that entered OCT probe 122 is reflected off of thetissue of subject 121 and nanoparticles 123. The reflected light energypasses back through the OCT probe 122 into the splitter 119 via Port 3.The reflected light energy that is returned into Port 2 and Port 3 ofthe splitter 119 recombines and interferes according to a split ratio.The light recombines either constructively or destructively, dependingon the difference of pathlengths. A series of constructive anddestructive combinations of reflected light can be used to create aninterferogram (a plot of detector response as a function of optical pathlength difference (cτ) or optical time-delay (τ)). Each reflectinginterface from the subject 121 and the nanoparticles can generate aninterferogram. The splitter 119 can recombine light energy that isreturned through Port 2 and Port 3 so that the light energies interfere.The light energy is recombined in the reverse of the split ratio. Forexample, if a 60/40 split ratio, only 40% of the light energy returnedthrough Port 2 and 60% of the light energy returned through Port 3 wouldbe recombined. The recombined reflected light energy is directed outPort 4 of the splitter 119 into a coupling lens 137. The coupling lens124 receives light from the output of the splitter 119 and sets the beametendue (beam diameter and divergence) to match that of the opticalspectrometer 125. The coupling lens 124 couples the light into anoptical spectrometer 125. The optical spectrometer 125 can divide therecombined reflected light energy light into different opticalfrequencies and direct them to different points in space which aredetected by a line scan camera 126. The line scan camera 126 performslight to electrical transduction resulting in digital light signal data108. The digital light signal data 108 is transferred into the computer101 via the OCT Input interface 111. Interface between the line scancamera 126 and computer 101 can be, for example, IEEE-488, IEEE-1394,Universal Serial Bus (USB), or the like. The digital light signal data108 can be stored in the mass storage device 104 or system memory 112and utilized by the image construction software 106 and the nanoparticlemovement (or cellular membrane tension level or internal strain fieldchange) image construction software 107.

The preceding exemplary phase sensitive OCT system is only one exampleof the contemplated systems for imaging tissues and nanoparticles.Variations in layout and equipment known to one skilled in the art arealso contemplated.

FIG. 18 is an exemplary block diagram of a Multi-Channel Phase SensitiveOCT system 2000. The exemplary Multi-Channel Phase Sensitive OCT system2000 can include a general-purpose computing device in the form of thecomputer 101, and all subsystems of the computer 101, as describedherein. The exemplary multi-channel Phase Sensitive OCT system 2000 canalso include, as previously described, a display device 127, a magnetcontrol 114, and a magnet 114 or a magnet 115.

Light energy is generated by a light source 2120. The light source 212can be a narrow band tunable laser light source wherein the opticalwavelengths generated range from about 400 nanometers to about 1600nanometers. Appropriate selection of a range of optical wavelengths canbe readily determined by one skilled in the art. For example, if lightenergy is to go through substantial water path, i.e., deep tissue, thenan operator can select longer optical wavelengths. For example,1300-1600 nanometers. The light spectrum is continuously varied in time,over a specified spectral region. A fraction of the light energy passesfrom the light source 2120 into an optical spectrum analyzer 118. Theoptical spectrum analyzer 118 samples a portion of the light emitted bythe light source 212. The optical spectrum analyzer 111 monitors thepower spectral density of light entering the circulator 2010. Theoptical spectrum analyzer 118 can measure optical frequency as it isemitted from the light source 2120 as a function of time. The remainingfraction of light energy generated by the light source 2120 passes intoa fiber circulator 2010. The fiber circulator 201 can comprise threeports, designated Port 1, Port 2, and Port 3. Light energy can enterPort 1. Light energy can exit and re-enter Port 2. Light energy can exitPort 3. The fiber circulator 2010 can recombine light energy thatre-enters via Port 2. Light energy from the light source 2120 passesinto the fiber circulator 2010 through Port 1. The light energy exitsthe fiber circulator 2010 through Port 2 and enters an OCT probe 2070.The light energy is coupled to a collimator lens 2020. The collimatorlens 2020 focuses the light emitted from the fiber at a point infinitelyfar from the fiber tip.

The light energy is collimated into a lens array 2030. The lens array2030 can comprise a lattice of microlenses or lenslets. The number ofmicrolenses in the lens array 2030 can be readily determined by oneskilled in the art. For each microlens, there is a fiber channel 204that is coupled to the microlens. Fiber channels 2040 are opticalwaveguides that confine and guide light along a path. The fiber channels2040 can be varied in length. Choosing an appropriate length for thefiber channels 2040 is known to one skilled in the art. The differencein the length between fiber channels 2040 can be from about one and ahalf to about ten times the scan depth in the tissue of a subject 121.This variable length can allow demultiplexing light signal detected fromthe channels. A fraction of the light energy transmitted into the fiberchannels 2040 is reflected from a reference reflector surface 120 backinto the fiber channels 2040, through the lens array 2030, into thecollimator lens 2020 and into the fiber circulator 2010. This reflectedlight energy can serve as a reference reflection. The light energy thatis not reflected back from the reference reflector surface 120 passesthrough the reference reflector surface 120 and onto an imaging lens2050. The imaging lens 2050 images the light energy from the tips of thefiber channels 2040 onto the tissue of the subject 121. The light energypasses through the imaging lens 2050 onto a reflector surface 2060,which turns the light energy 90 degrees. This allows the light energy tobe reflected out radially inside a tissue. There is one reflectorsurface 2060 for each fiber channel 2040. The light energy that isturned 90 degrees by the reflector surface 2060 is back reflected off ofthe tissue of subject 121, and nanoparticles 123.

The light is reflected from the tissue of subject 121 and thenanoparticles 123. The light energy strikes the reflector surface 2060and is turned back 90 degrees. The light energy is then coupled by theimaging lens 2050 through the reference reflector surface 120 and backinto each fiber channel 2040. The light energy reflected from thenanoparticles 123 and the tissue of subject 121 recombines andinterferes with the light reflected from the reference reflector surface120 in the fiber channels 2040. The recombined light energy can becoupled back into the lens array 2030 through the collimator lens 2020and back into Port 2 of the fiber circulator 2010. The recombined lightenergy exits the fiber circulator 2010 through Port 3. A coupling lens2080 couples the recombined light energy from the fiber circulator 2010into a photo receiver 2090. The photo receiver 2090 converts the lightenergy signal into a voltage signal that is proportional to the numberof photons contained in the recombined light energy. The voltage signalpasses from the photo receiver 2090 into a pre/amp 2100. The pre/amp2100 takes the voltage signal and amplifies it. The amplified voltagesignal enters an A/D converter 2110. The A/D converter 2110 digitizesthe voltage signal. This digital light signal data then enters thecomputer 101 through the OCT input interface 111. The digital lightsignal data 108 can be stored in the mass storage device 104 or systemmemory 112 and utilized by the image construction software 106 and thenanoparticle movement (or cellular membrane tension level or internalstrain field) image construction software 107.

The method can further comprise generating light energy for at least twosuccessive sweeps of light energy. A sweep is an emission of light froma light source across a range of optical frequencies. Multiple sweepscan be combined with application of a magnetic field to generate imageswith and without a magnetic field applied.

The method can further comprise applying a magnetic field to the subjectfor each of the successive sweeps of the light energy wherein thestrength of the magnetic field applied in a sweep is greater than thestrength of the magnetic field from the preceding sweep and wherein themagnetic field causes movement of at least one of the metallicnanoparticles. The method can further comprise applying the magneticfield from a source external to the subject or from a source internal tothe subject. A coil generating the magnetic field can be integrated intoa catheter or can be external to the subject of the scan.

As shown in FIG. 18, a non-uniform magnetic field can be applied to thetissue of subject 121 and the nanoparticles 123. The non-uniformmagnetic field can be applied by the magnet 116, which can be a magnetinternal to the OCT probe 122 or the non-uniform magnetic field can beapplied externally to the subject 121 by a magnet. Magnets 116 andexternal magnet are both controlled by magnet control 114. The magnetcontrol can provide the current source to power external magnet andmagnet 116 and is under the control of the computer 101. The magnetcontrol 114 interfaces with the computer 101 through the OCT outputinterface 110. The magnet control 114 can interface with the computer101 via IEEE-488, IEEE-1394, Universal Serial Bus (USB), or the like.

The method can further comprise processing the received light energy toproduce a phase sensitive OCT image. The image produced can have a phaseresolution of at least 30 nanometers (nm). Phase resolution is definedas the phase delay of the light signal returning from the tissuescanned. For example, the image can have a phase resolution of about atleast 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5 ml, 4 nm, 3 nm, or 2 nm.

The processing of the received light energy can be performed by softwarecomponents. The image construction software 106 and the nanoparticlemovement (or cellular membrane tension level or internal strain field)image construction software 107 can be described in the general contextof computer-executable instructions, such as program modules, beingexecuted by one or more computers or other devices. Generally, programmodules include computer code, routines, programs, objects, components,data structures, etc. that perform particular tasks or implementparticular abstract data types. The image construction software 106 andthe nanoparticle movement (or cellular membrane tension level orinternal strain field) image construction software 107 can also bepracticed in distributed computing environments where tasks areperformed by remote processing devices that are linked through acommunications network. In a distributed computing environment, programmodules can be located in both local and remote computer storage mediaincluding memory storage devices.

The image construction software 106 can generate an image of the tissueof subject 121 from the light signal data 108. The image constructionsoftware 106 can receive the light signal data 108 and can perform atime-frequency transform (e.g. Fourier transform) on the light signaldata 108 generating amplitude and phase data. The amplitude and phasedata (optical path length difference (cτ) or optical time-delay (τ)) canbe separated into discrete channels and a plot of intensity vs. depth(or amplitude vs. depth) can be generated for each channel. Such a plotis known in the art as an “A” scan. The composition of all the “A” scanscan comprise one image.

The nanoparticle movement (or cellular membrane tension level orinternal strain field) image construction software 107 generates animage of the movement of the nanoparticles 123 from the light signaldata 108. The nanoparticle movement (or cellular membrane tension levelor internal strain field) image construction software 107 receives thelight signal data 108 for at least two successive sweeps of the lightsource 117 or the light source 212 and performs a Fourier transform onthe light signal data 108 generating amplitude and phase data. Theamplitude and phase data can be separated into discrete channels, onechannel for each fiber channel 204, and a plot of phase vs. depth(optical time-delay (τ)) can be generated for each channel. Points ofnanoparticle 123 movements are identified by the phase changes betweentwo successive sweeps of the light source 117 or the light source 212corresponding to two applied magnetic field strengths.

Optionally, an optical clock is used to trigger acquisition of thesignal produced by the photodetector. The optical clock provides a setof uniformly spaced clock pulses with fixed intervals of opticalfrequency and at least one reference pulse. The fixed intervals ofoptical frequency are configured and specified in the optical clock togive a uniform train of pulses. The at least one reference pulsegenerated by the optical clock is utilized to provide a referenceoptical frequency or a trigger pulse. For example, the first referencepulse generated by the optical clock can correspond to an absorptionline in a gas cell (e.g., Hydrogen Fluoride or Hydrogen Bromide). Inthis case the gas absorption line has a known optical frequency. Thewell-known absorption fingerprint bands in the HF gas cell result in areduced detected intensity in the light transmitted through the gascell, and as such provide a metric on the absolute lasing wavelength atthe digitized samples of the photodetector signal. The digitized samplenumber or sampling time scale can thus be converted to absolutewavelength at one or more samples, depending on the number of absorptionlines. The detected wavemeter photocurrent signal and the detected gascell photocurrent signal are combined in the digitizer to provide therelationship between the sample number or sampling time and lasingwavelength throughout the entire sweep. The detected photocurrent signalfrom the gas cell is digitized concurrently with the OCT interferogramand correlated with the known HF fingerprint to determine the wavenumberbias (k_(o)) of the swept source laser. Knowledge of wavenumber bias(k_(o)) allows accurate determination of the absolute wavenumber of eachdigitized sample throughout the spectral sweep, effectively removing anywavenumber offsets and/or phase instabilities in the laser source,wavemeter and sampling electronics. Knowledge of the magnitude of thefixed intervals and the optical frequency of at least one clock pulseprovides knowledge of the optical frequency of every clock pulseprovided by the optical clock.

Optionally, additional information can be extracted from the lightsignal data 108 to generate additional images. The light signal data 108can be further processed to extract the Doppler frequency shift as isreadily known to one skilled in the art. The light signal data 108 canalso be further processed to generate a Stokes parameter polarimetricimage when used in conjunction with polarization sensitive OCT toextract polarization data from the light signal 108 as is readily knownto one skilled in the art.

The methods and systems can be used to perform molecular identificationto stabilize vulnerable plaque that is anticipated to rupture and causeheart attacks, strokes, and progression of peripheral vascular disease.It can also be used to identify macrophages present in degenerative eyediseases which can lead to blindness and selectively kill thesepathologic cells to stabilize the eye degeneration. Generally, manycancers are known to be associated with the presence of macrophages, andit may provide a method for early cancer detection and treatment.

Example 2

A solenoid coil with a ferrite core was used to apply a sinusoidalmagnetic field to tissues taken from the liver of an ApoE−/− knockoutmouse. One mouse was loaded with magnetic nanoparticles one week beforeimaging while an unloaded mouse served as a control. FIG. 11A shows asolenoid drive signal (top) and optical pathlength change (bottom)observed in mouse loaded with nanoparticles. FIG. 11B shows a solenoiddrive signal (top) and optical pathlength change (bottom) observed incontrol mouse (no nanoparticles). These data demonstrate that iron oxideparticles that have been ingested by macrophages in livers and spleensof the mice. Moreover, the particles have put in motion with a magnetand detected with differential phase OCT using the systems and methodsdescribed herein.

To calculate magnetic field strength a finite element method (FEM) canbe used. Maxwell equations subjected to certain boundary conditions canbe used to solve low-frequency magnetostatic problems. The use andsolution of Maxwell equations are described in, for example, Monk P.,Finite Element Methods for Maxwell's Equations, Oxford University Press,2003, which is incorporated in its entirety by reference.

Maxwell equations can be written as:

$\begin{matrix}{{\nabla{H}} = {J + \frac{\partial D}{\partial t}}} & (1.1) \\{{\nabla{E}} = {- \frac{\partial B}{\partial t}}} & (1.2) \\{{\nabla{\cdot H}} = \rho} & (1.3) \\{{\nabla{\cdot B}} = 0} & (1.4)\end{matrix}$

In case of magnetostatic problems

$\left( {\frac{\partial D}{\partial t} = 0} \right),$

the magnetic field (H) and magnetic flux density (B) are satisfied withfollowing equations:

∇×H=J  (1.5)

∇·B=0  (1.6)

B and H are subject to a generalized constitutive relation:

B=u ₀(H+M)  (1.7)

Finite element methods (FEM) used magnetic vector potential (A) to findmagnetic field strength.

B=∇×A  (1.8)

Equation (1.5) can be rewritten as

∇×(u ₀ ⁻¹ ∇×A−M)= J  (1.9)

From equation (1.9), magnetic field strength and flux density can besolved. The symbols and unit for electromagnetic quantities for solvingFEM problems include:

-   -   H: Magnetic field (Ampere/m)    -   E: Electric field (Volt/meter)    -   B: Magnetic flux density (Tesla)    -   D: Electric flux density (Coulomb/meter²)    -   A: Magnetic potential (Weber/meter)    -   M: Magnetization (Ampere/meter)    -   u₀:Permeability of vacuum=4Π·10 ⁻⁷ (H/m)

${\nabla B} = {{divB} = {{\frac{\partial}{\partial x}B_{x}} + {\frac{\partial}{\partial y}B_{y}} + {\frac{\partial}{\partial z}B_{z}}}}$

is divergence of B)

Magnetic fields of between about 0.5, 1.5 and 2.0 Tesla were used tocause movement of the nanoparticles. Magnetic fields between about 1.0and 9.0 Tesla can also be used. The magnetic field used is typicallyhigher if the tissue of interest comprises a greater number ofnanoparticles or iron, when compared to tissue with fewer nanoparticlesor iron.

Example 3

Colloidal suspensions of SPIO nanoparticles are tissue-specific MRIcontrast agents approved by the United States Food and DrugAdministration (FDA) for human use in 1997. SPIO particles are alsoknown as Ferumoxides or AMI-25 and their trade name is Feridex® I.V.(USA) and Endorem® (EU). Mean core diameter of these particles is 20 nmand total aggregation diameter is about 100 nm. SPIO nanoparticlescomprise nonstoichiometric magnetite crystalline cores, iron, anddextran T-10 coating that is used to prevent aggregation andstabilization in the liver. 80% of injected dose of SPIO nanoparticlesaccumulate in tissue based macrophages (Kupffer cells) due to therelatively short blood half life compared to ultrasmall SPIOnanoparticles. Uptake of SPIO nanoparticles by macrophage cells isdirectly proportional to the intravenous injection (IV) concentration,blood half life, and core size.

To evaluate magnetic force on superparamagnetic (SPIO) nanoparticles,magnetic potential energy, U, can be used to calculate force due toapplication of an external magnetic flux density (B).

$\begin{matrix}{U = {{- \frac{1}{2}}{m \cdot B}}} & (11)\end{matrix}$

If a magnetic material is exposed to an external magnetic flux density,B, the individual particles have overall response determined by themagnetic moment, m. The magnetic flux density on magnetic nanoparticlescan be written:

B=u ₀(H+M)  (12)

where μ₀ (4π×10⁻⁷ H/M) is the permeability of free space, and M is themagnetic moment per unit volume and H is magnetic field strength. Themagnetic moment, m, acting on magnetic volume, V is given by, m=M V.Magnetization of magnetic particles can be classified in terms of thestandard relation M=χH. Therefore, magnetic moment m becomes:

m=MV=χ _(s) VH=χ _(s) VB/u ₀  (13)

In Eq. (13), susceptibility of the SPIO particles χ_(s) is dimensionlessin SI units and given by dipole density for each paramagnetic materialand is an important parameter characterizing magnetic properties of SPIOnanoparticles. From Eq. (11), magnetic energy U, of a SPIO nanoparticlesin external magnetic field is given by,

$\quad\begin{matrix}\begin{matrix}{U = {{- \frac{1}{2}}{m \cdot B}}} \\{= {{- \frac{\chi \; V}{2u_{0}}}B^{2}}}\end{matrix} & (14)\end{matrix}$

Magnetic force acting on SPIO nanoparticles becomes:

$\quad\begin{matrix}\begin{matrix}{F = {- {\nabla U}}} \\{= {\nabla\left( {\frac{\chi_{s}V}{2u_{0}}B^{2}} \right)}} \\{= {\chi_{s}V{{\nabla\left( \frac{B^{2}}{2u_{0}} \right)}.}}}\end{matrix} & (15)\end{matrix}$

A sinusoidal magnetic flux density that is principally along thez-direction was assumed. Hence, {right arrow over (B)}(x, y, z; t)sin(2πf_(n)t)B_(z)(z){circumflex over (k)} and the magnetic force F_(z)acting on nanoparticles in the z-direction is given by

$\begin{matrix}\begin{matrix}{{\sum F_{z}} = {m\frac{\partial^{2}{z(t)}}{\partial t^{2}}}} \\{= {F_{m} - {{kz}(t)} - {r\frac{\partial z}{\partial t}}}}\end{matrix} & (16) \\{{{\sum F_{z}} = {{{\frac{\chi_{s}V_{s}}{2\mu_{0}}\left\lbrack {1 - {\cos \left( {4\pi \; f_{n}t} \right)}} \right\rbrack}{B_{z}(z)}\frac{\partial B_{z}}{\partial z}} - {{kz}(t)} - {r\frac{\partial z}{\partial t}}}},} & (17)\end{matrix}$

where F_(m) is magnetic force, f_(n) is the modulation frequency of theapplied sinusoidal magnetic field, kz(t) is an elastic restoring force,and

$r\frac{\partial z}{\partial t}$

is a viscous drag force that account for the viscoelastic properties ofthe local tissue environment. The negative sign of the viscous drag andrestoring force indicates that this force is in opposite direction tomovement z(t). Equation 17 can be written by dividing by the mass, m.

$\begin{matrix}{{\frac{\partial^{2}{z(t)}}{\partial t^{2}} + \frac{{kz}(t)}{m} + {\frac{r}{m}\frac{\partial z}{\partial t}}} = {{\frac{\chi_{s}V_{s}}{2m\; \mu_{0}}\left\lbrack {1 - {\cos \left( {4\pi \; f_{n}t} \right)}} \right\rbrack}{B_{z}(z)}\frac{\partial B_{z}}{\partial z}}} & (18)\end{matrix}$

Equation 18 can be rewritten using the first terms in the Maclarinseries for the magnetic field,

$\begin{matrix}{{\frac{\partial^{2}{z(t)}}{\partial t^{2}} + {\frac{r}{m}\frac{\partial z}{\partial t}} + \frac{{kz}(t)}{m}} \cong {{\frac{\chi_{s}V_{s}}{2m\; \mu_{0}}\left\lbrack {1 - {\cos \left( {4\pi \; f_{n}t} \right)}} \right\rbrack}{B_{z}(0)}\frac{\partial{B_{z}(0)}}{\partial z}}} & (19)\end{matrix}$

Letting

${a = {\frac{\chi_{s}V_{s}}{2m\; \mu_{0}}{B_{z}(0)}\frac{\partial{B_{z}(0)}}{\partial z}}},{c = {4\pi \; f_{n}}},$

the second order differential Eq. (19) can be written

$\begin{matrix}{{{\frac{\partial^{2}{z(t)}}{\partial t^{2}} + {\frac{r}{m}\frac{\partial z}{\partial t}} + \frac{{kz}(t)}{m}} = {a\left\lbrack {1 - {\cos ({ct})}} \right\rbrack}},} & (20)\end{matrix}$

The Laplace transform can be used to solve the second order differentialequation (20), assuming zero initial displacements and velocity to find;

$\begin{matrix}{{{{s^{2}{Z(s)}} + {\frac{r}{m}{{sZ}(s)}} + {\frac{k}{m}{Z(s)}}} = {\frac{a}{s} - \frac{as}{\left( {s^{2} + c^{2}} \right)}}}{{Z(s)} = {\frac{\frac{a}{s} - \frac{as}{\left( {s^{2} + c^{2}} \right)}}{\left( {s^{2} + {\frac{r}{m}s} + \frac{k}{m}} \right)} = {a\left( {\frac{1}{\left( {s^{2} + {\frac{r}{m}s} + \frac{k}{m}} \right)s} - \frac{s}{\left( {s^{2} + {\frac{r}{m}s} + \frac{k}{m}} \right)}} \right)}}}} & (21)\end{matrix}$

By computing the sum of the transforms, Z(s) can be derived in Eq. (21)

$\begin{matrix}{{z(t)} = \frac{{ma}\begin{pmatrix}\begin{matrix}{{{- 2}{mkc}^{2}} + {c^{2}r^{2}} + k^{2} + {c^{4}m^{2}} -} \\{{\cos ({ct})k^{2}} + {{\cos ({ct})}c^{2}{mk}} - {{cr}\; {\sin ({ct})}k} +}\end{matrix} \\{{\exp \left( {{- \frac{1}{2}}\frac{tr}{m}} \right)}\begin{pmatrix}{c^{2}\left( {{km} - r^{2} - {c^{2}m^{2}\cosh}} \right.} \\{\left( {\frac{1}{2}\frac{{t\left( {r^{2} - {4{km}}} \right)}^{\frac{1}{2}}}{m}} \right) +} \\\frac{\begin{matrix}{c^{2}{r\left( {{3{km}} - r^{2} - {c^{2}m^{2}}} \right)}} \\{\sinh \left( {\frac{1}{2}\frac{{t\left( {r^{2} - {4{km}}} \right)}^{\frac{1}{2}}}{m}} \right)}\end{matrix}}{\left( {r^{2} - {4{km}}} \right)^{\frac{1}{2}}}\end{pmatrix}}\end{pmatrix}}{\left( {k\left( {{{- 2}{mkc}^{2}} + {c^{2}r^{2}} + k^{2} + {c^{4}m^{2}}} \right)} \right.}} & (22)\end{matrix}$

The displacement z(t) of nanoparticles can be found by using an inverseLaplace transform; the solution includes transient and steady stateterms. The initial motion of magnetic nanoparticles is driven by aconstant magnetic force and displays a damped transient motion beforesteady state motion dominates at twice the modulation frequency (f_(n))of the applied sinusoidal magnetic field. Motion of the nanoparticles atdouble the modulation frequency originates from the magnetic force beingproportional to the product of the field and field-gradient (Eq. 17).

Liver tissues from 12 week old ApoE^(−/−) high fat fed mice wereutilized because they contain tissue based macrophages cells. The micewere injected via the jugular vein with either Feridex I.V. (Ferumoxidesinjectable solutions; Berelex Laboratories, Montville, N.J.) forintravenous administration (1.0, 0.1, and 0.01 mmol Fe/kg body weight)or saline and sacrificed 2 days post intravenous injection. The micewere euthanized with a lethal dose of Ketamine and Xylazine. Aftereuthanizing, abdominal incisions were made to remove the entire liverfrom the mouse. Portions were cut using a microtome. Physical thicknessof the liver samples was 1 mm and 0.5 cm×0.5 cm in lateral dimensions.After completion of the DP-OCT measurements, the mouse livers wereembedded in 10% formalin acid, and processed for histology. 5 μm thicksections were cut and stained with Prussian blue to identify irondeposition in liver Kupffer cells in mouse liver tissues. To verify SPIOuptake by macrophage cells from histology slides, Image Pro Plus®(Mediacynernetics Inc., Silver Spring, Md.) was used to measure thetotal area of liver and accumulated area of SPIO aggregation containingPrussian blue positive.

FIGS. 12A and 12B shows a schematic diagram of a fiber-based dualchannel differential phase optical coherence tomography (DP-OCT) system(a), and sample path configuration with a magnetic field generator (b).The magnetic field generator comprises a solenoid, signal generator andcurrent amplifier. A dual-channel Michelson interferometer was used tomeasure differential phase between light backscattered from a sample byapplying a sinusoidal focused magnetic field excitation. Partiallypolarized light from an optical semiconductor amplifier (AFCTechnologies, Rancho Cordova, Calif., central wavelength λ₀=1.31 μm,FWHM=60 nm, optical coherence length=22 μm) is polarized and coupledinto fast and slow axes of a polarization-maintaining (PM) fiber in theinput port.

Optical path length change (Δp) in tissue can be calculated from thedifferential phase (Δφ) and central wavelength of a broad-band lightsource (λ₀=1,310 nm) between the two channels.

$\begin{matrix}{{\Delta \; p} = {\frac{\lambda_{0}}{4\pi}\Delta \; \phi}} & (23)\end{matrix}$

The displacement z(t) of tissue-laden nanoparticles driven by a time (t)varying magnetic flux density can be derived the analytic OCT fringeexpression,

$\begin{matrix}{I_{f} = {2\sqrt{I_{R}I_{S}}{\cos \left\lbrack {{2\pi \; f_{0}t} + \frac{4\pi \; {z(t)}}{\lambda_{0}}} \right\rbrack}}} & (24)\end{matrix}$

Where I_(R) and I_(s) are the back scattered signals from the referenceand sample arms, respectively. f₀ is the fringe carrier frequency, andz(t) is the nanoparticles displacement. The OCT fringe signal can beexpressed by the nanoparticles displacement equation (24). The twosignals recorded from Channel 1 and 2 by the DP-OCT system can be usedto measure nanoparticles displacement that represent relative surfacetissue displacement between two scanning beams.

Finite element method (FEM) was used to design the magnetic fieldgenerator and evaluate space-time magnetic flux density. The magneticfield generator comprises a solenoid (Ledex 6EC, Saia-Burgess Inc.,Vernon Hills, Ill.), a function generator (HP 33120A, Hewlett PackardInc., Palo Alto, Calif.), a current amplifier, and a power supply. FEMcalculations (Maxwell S V, Ansoft Inc., Pittsburgh, Pa.) and Teslameter®(Magnetometer®, AlphaLab Inc., Salt Lake City, Utah) measurementindicated that the maximum magnetic flux density at a distance of 1.5 mmfrom the tip of the iron core was approximately 2 Tesla. The FEMsimulation demonstrated that an iron core positioned along thecenterline of the solenoid dramatically increased magnetic flux densityat the target specimen. Magnetic field distributions from the FEMsimulation showed the maximal and principal direction of the magneticfield strength was in the z-direction. The conical iron core providedfocusing and substantially increased the magnetic field strength.

Differential phase OCT (DP-OCT) measurements were performed on isolatedliver specimens taken from ApoE−/− mice administrated with differentSPIO doses (1.0, 0.1 and 0.01 mmol Fe/kg body weight) and saline controlsamples. FIG. 7 demonstrates measurements of transient optical pathlength change (Δp) in specimens at different SPIO doses (1.0, 0.1 mmolFe/kg body weight) and saline control samples, in response toapplication of a sinusoidal varying focused magnetic field. FIG. 13Ashows a magnetic field input (f_(n)=2 Hz), peak-to-peak voltage(V_(pp)=4) over a 1 second time period. The maximum magnetic fieldstrength was 0.47 Tesla and maximal tissue displacement by optical pathlength change (Δp) was 2,273 nm in the 1.0 mmol Fe/kg iron-laden liver.Compared to high dose specimens, 0.1 mmol Fe/kg iron-laden liver showeda maximum optical path length change (Δp) of 127 nm with additive noisevisible in recorded signals. Frequency response (4 Hz) of iron-ladenlivers (FIG. 7 (b), (c)) was exactly twice the modulation frequency (2Hz) as noted earlier. No significant displacement of SPIO nanoparticleswas observed in either saline control liver specimens in the FIG. 13D orsamples at the 0.01 mmol Fe/kg dose (not shown).

SPIO nanoparticle movement in the iron laden livers (0.1, and 1.0 mmolFe/kg) was used to observe quantitatively the relationship betweenoptical path length change (Δp) versus different applied magnetic fieldstrengths, as shown in FIG. 14A. Input frequency used in this experimentwas 2 Hz with amplitude from 2 to 8 V_(pp). FIG. 8 (b) shows magneticflux density at the same voltages as in FIG. 14A. Magnitude of opticalpath length change (Δp)) indicating movement of iron-laden liverdepended directly on the SPIO dose concentration, and strength of theexternal magnetic field.

Optical path length change (Δp) at high frequency modulation (over 100Hz) was negligible due to limited frequency response of the structuressurrounding SPIO nanoparticles. Generally, optical path length change(Δp) due to nanoparticles movement in tissue increased with highermagnetic field strength.

Optical path length change (Δp) in the iron-laden liver (0.1 and 1.0mmol Fe/kg) can be measured using a swept input frequency as shown inFIG. 15A. FIG. 15A shows the magnetic field input with a swept frequencyfrom 1 to 10 Hz over a 2 second time-period. Magnitude of the opticalpath length change (Δp) was 2,318 nm in a high dose liver (1.0 mmolFe/kg) and 177 nm in a low dose concentration (0.1 mmol Fe/kg), andmagnetic field strength was 1.3 Tesla. The frequency response of theforce acting on the iron-laden liver is exactly twice the externallyapplied modulated frequency in FIG. 15B and FIG. 15C. No significantdisplacement was observed in the saline control liver shown in FIG. 9(d) and 0.01 mmol Fe/kg liver specimens.

FIGS. 16A and 16B illustrates SPIO nanoparticle movement measured byoptical path length change (Δp) in a iron-laden mouse liver to observequantitatively the relationship between magnetic response in tissueversus different applied magnetic field strengths with swept frequencyranging from 1˜10 Hz over a 2 second time-period. Magnitude of opticalpath length change (Δp) was larger when input voltage was graduallyincreased from 2 to 10 V_(pp) during a frequency sweep. Correspondingmagnetic field strength at these voltages was 1.24, 1.58, 1.71, 1.75 and1.84 Tesla, respectively. For a given frequency sweep, maximum opticalpath length change (Δp) for 0.1 and 1.0 mmol Fe/kg iron-laden liverspecimens was 3,700 nm and 750 nm, respectively, at 10 V_(pp), andmagnetic field of 1.84 Tesla.

SPIO nanoparticles were identified in histological specimens as bluegranules from the Prussian blue stain of iron laden mouse livers.Compared to control liver specimens, iron laden specimens showsignificant iron accumulation evenly distributed in all observed areas.Although intracellular iron was also observed in control specimens, thisnatural iron was uniform and homogeneous rather than appearing ingranular shapes as SPIO iron nanoparticles. Total SPIO iron area was5.45% of the histology image as calculated by Image-Pro PLUS 5.1software (Mediacynernetics Inc., Silver Spring, Md.).

Example 4

Optical path length change (Δp) in iron-laden rabbit arteries (0.1Fe/kg) was measured in response to 2 Hz frequency sinusoidal input FIG.17. FIG. 17A shows the magnetic field input with a constant frequency at2 Hz over a 2.5 second time-period. Magnitude of the optical path lengthchange (Δp) indicated a transient and steady state response. Transientresponse is evident in the exponentially decaying oscillation in theobserved measured optical path length change at times between 0.5-1.0seconds. Steady state response is evident in the uniform oscillation inthe measured optical path length change at times between 1.25-3.0seconds. Transient response indicates a high frequency (40 Hz-80 Hz)“ringing” oscillation and a damping relaxation time of approximately 0.3seconds. The steady state frequency response of the force acting on theiron-laden rabbit artery was exactly twice the externally appliedmodulated frequency in FIG. 11 (b).

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

While the invention has been described in connection with variousembodiments, it will be understood that the invention is capable offurther modifications. This application is intended to cover anyvariations, uses or adaptations of the invention following, in general,the principles of the invention, and including such departures from thepresent disclosure as, within the known and customary practice withinthe art to which the invention pertains.

1. A system for imaging, comprising: a magnet for applying a magneticfield to the vessels to be imaged, wherein the applied magnetic fieldinteracts with hemoglobin within the vasculature; and an opticalcoherence tomography apparatus for detecting magnetic movement of thehemoglobin within the vasculature.
 2. The system of claim 1, wherein themagnetic field is oscillating.
 3. The system of claim 1, wherein themagnet comprises a solenoid coil with a ferrite core.
 4. The system ofclaim 3, wherein the optical coherence tomography apparatus furthercomprises a sample arm, and wherein a probe is coupled to the sample armof the optical coherence tomography apparatus and the magnet.
 5. Thesystem of claim 4, wherein the optical coherence tomography apparatus isa magneto-motive optical Doppler tomography imaging system for detectingthe blood flow affected by the magnetic field.
 6. The system of claim 5,wherein the magneto-motive optical Doppler tomography system comprises:a light source generating light energy; an interferometer coupled to thelight energy, wherein the interferometer includes a reference and samplelight paths coupled to a light splitter; a modulator coupled to theinterferometer for modulating the optical path length difference in thereference arm and the sample arm; a scanner coupled to the sample armfor scanning a biological sample; a rapid scanning optical delay linecoupled to the reference arm; a photodetector coupled to theinterferometer for detecting backscattered radiation received by theinterferometer from the scanner to detect interference fringes; and aprocessor for processing the reflected light energy from the referencearm and a signal reflected off of the moving blood flow to produce atomographic image and a tomographic flow velocity image.
 7. The systemof claim 6, wherein the magneto-motive optical Doppler tomography systemincludes a scanning element to permit three dimensional scans.
 8. Thesystem of claim 7, wherein the magneto-motive optical Doppler tomographysystem includes a dual-balanced photodetector.
 9. The system of claim 8,wherein the magneto-motive optical Doppler tomography system includes acirculator coupled to the interferometer and the photodetector.
 10. Amethod for imaging a blood flow, comprising: applying a magnetic fieldto the blood flow, wherein the blood flow comprises a plurality ofhemoglobin molecules and wherein the magnetic field interacts with thehemoglobin to cause a change in the blood flow; and detecting the bloodflow by detecting the change in the blood flow caused by the interactionwith the hemoglobin molecules with the magnetic field, wherein thechange is detected using a optical coherence tomography system.
 11. Themethod of claim 10, wherein the applying of the magnetic field comprisestemporally oscillating the magnetic field.
 12. The method of claim 11,further comprising coupling the optical coherence tomography system andthe magnetic field to a probe.
 13. The method of claim 12, wherein thedetecting the blood flow by detecting the change in the blood flowcaused by the interaction with the hemoglobin molecules, wherein thechange is detected using a magnetomotive optical Doppler tomographyimaging system.
 14. The method of claim 13, wherein the magneto-motiveoptical Doppler tomography system comprises the method of providinglight energy through an interferometer; phase modulating the lightenergy in the interferometer at a modulation frequency; continuouslyscanning a blood flow sample with the light energy through theinterferometer, wherein the blood flow sample includes a blood flowtherein and a structure in which the blood flow is defined; detectingthe signal reflected off the moving blood sample and the interferencefringes of the light energy backscattered from moving blood sample; anddata processing Doppler frequency changes of the detected backscatteredinterference fringes with respect to said modulation frequency at eachpixel of a scanned image to continuously measure the interference fringeintensities to obtain time dependent power spectra for each pixellocation in a data window in a continuous scan from which a tomographicimage of the blood flow in and the structure of said scanned blood flowsample is formed.
 15. The method of claim 13, wherein the modulationfrequency is zero.
 16. The method of claim 14, where detecting theinterference fringes of light energy backscattered from the moving bloodsample includes reducing the light source noise from the interferencesignal with a dual balanced photodetector.
 17. The method of claim 15,further including improving imaging speed with a hardware in-phase and aquadrature demodulator with at least one high-bandpass filter.
 18. Theapparatus of claim 4, wherein the optical coherence tomography systemfor detecting blood flow is a spectral domain phase sensitive opticalcoherence tomography system.
 19. The apparatus of claim 4, wherein thephase sensitive optical coherence tomographic imaging modality is aswept source phase sensitive optical coherence tomography systemincluding a tunable swept source laser and an optical clock to sampleinterference fringe data uniformly spaced in the optical frequencydomain.
 20. The method of claim 10, wherein the detecting of the changein the blood flow caused by the interaction with the hemoglobinmolecules is detected using a spectral domain phase sensitive opticalcoherence tomography system.
 21. The method of claim 14, wherein thedetecting of the change in the blood flow caused by the interaction withthe hemoglobin molecules is detected using a swept source phasesensitive optical coherence tomography system.
 22. An apparatus forimaging a blood flow comprising: a magnetic field generator for applyinga magnetic field to the blood flow, wherein the blood flow compriseshemoglobin molecules; and an ultrasound detection system for detectingthe blood flow while it is in the presence of the magnetic field. 23.The apparatus of claim 22, wherein the ultrasound detection systemcomprises a probe for transmitting and receiving sound energy to andfrom a subject and the probe includes the magnetic field generator.